Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02701198 2015-03-10
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CA2701198
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Sensitive And Rapid Methods Of Using Chimeric Receptors
To Identify Autoimmune Disease
Field
The present disclosure provides methods and compositions useful in the
diagnosis of
autoimmune diseases. In particular, the present disclosure provides methods
and compositions for
use in the diagnosis and management of Graves' disease. For example, one
composition comprises a
chimeric thyroid stimulating hormone receptor having improved sensitivity and
specificty for
circulating thyroid stimulating immunoglobulin. Assays using such chimeric
receptors can be
optimized in the presence of a glucocorticoid.
Background
Graves' disease (also referred to as "diffuse toxic goiter"), is the leading
cause of
hyperthyroidism due to the action of autoantibodies that recognize and bind to
receptors present on
the thyroid gland, resulting in gland growth and over-production of thyroid
hormone. Graves'
disease is reported to be the most frequent cause of hyperthyroidism in
childhood and adolescence
(See, Boter and Brown, J. Pediatr. 132:612-618 (1998)).
Current diagnostic techniques for Graves' disease leave much to be desired. In
general, the
commercially available methods are cumbersome and laborious. Other methods
require the
administration of radioactive tracers to the person requiring a diagnosis.
Most importantly,
however, the vast majority of the presently used methods lack sufficient
sensitivity such that a
quick, accurate and cost-effective test can be performed.
What is still needed is an assay system for Graves' disease that is safe, easy
to use, sensitive,
specific, and cost-effective.
Summary
The present disclosure provides methods and compositions useful in the
diagnosis and
management of autoimmune diseases. In particular, the present disclosure
provides methods and
compositions for the diagnosis and management of Graves' disease. For example,
one composition
comprises a chimeric thyroid stimulating hormone receptor having improved
sensitivity and
specificty for circulating thyroid stimulating immunoglobulin. Assays using
such chimeric
receptors can be optimized in the presence of a glucocorticoid.
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One embodiment contemplates a method, comprising: a) providing; i) a cell line
comprising
a stably transfected vector encoding a chimeric TSH receptor and a luciferase
gene; ii) a serum
sample derived from a patient suspected of having Graves' disease; and iii) a
cell culture medium
comprising a glucocorticoid; b) contacting the serum sample with the cell line
and the medium
under conditions such that the luciferase gene emits a detectable signal. In
one embodiment, the
method further comprises step c) measuring the signal intensity, wherein the
intensity correlates
with a thyrotropin stimulating hormone receptor autoantibody concentration
present in the sample.
In one embodiment, the glucocorticoid is selected from the group comprising
dexamethasone,
prednisone, hydrocortisone, fluticasone, or cortisone. In one embodiment, the
contacting further
comprises polyethylene glycol. In one embodiment, the chimeric TSH receptor
comprises an amino
acid sequence derived from rat chorionic hormone gonadotropin receptor. In one
embodiment, the
amino acid sequence comprises seventy three amino acids corresponding to amino
acid residues
262-335 of a human TSH receptor amino acid sequence. In one embodiment, the
serum sample
comprises TSH receptor autoantibodies. In one embodiment, the autoantibodies
comprise TSH
stimulating autoantibodies. In one embodiment, the autoantibodies comprise TSH
blocking
antibodies.
One embodiment contemplates a kit comprising a chimeric TSH receptor and a
luciferin-
luciferase system capable of detecting serum TSH autoantibodies, wherein the
system comprises a
glucocorticoid. In one embodiment, the glucocorticoid is selected from the
group comprising
dexamethasone, prednisone, hydrocortisone, fluticasone, or cortisone. In one
embodiment, the
receptor comprises a human TSH receptor amino acid sequence. In one
embodiment, the receptor
comprises a rat chorionic hormone receptor amino acid sequence. In one
embodiment, the rat
receptor amino acid sequence comprises amino acid residues 262-335. In one
embodiment, the kit
further comprises a cell line capable of expressing the chimeric TSH receptor
and the luciferin-
luciferase system. In one embodiment, the kit further comprises polyethylene
glycol. In one
embodiment, the kit comprises a vector encoding the chimeric TSH receptor and
a luciferase gene.
In one embodiment, the vector further comprises a promoter in operably linked
to the vector. In one
embodiment, the promoter comprises a glycoprotein alpha subunit promoter. In
one embodiment,
the cell line comprises CHO cells. In one embodiment, the cell line comprises
RD cells. In one
embodiment, the kit further comprises an instruction sheet.
One embodiment provides methods for determining the presence of thyroid-
stimulating
autoantibodies in a test sample, comprising: a) providing i) a test sample
suspected of containing
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thyroid-stimulating autoantibodies, ii) cultured cells comprising a
glucocorticoid contained within a
testing means, wherein the cells express a chimeric TSH receptor and a
luciferin-luciferase system,
and iii) polyethylene glycol; b) exposing the test sample to the cultured
cells and polyethylene
glycol under conditions such that thyroid-stimulating antibodies are
detectable using a luciferin-
luciferase system; and c) observing for the presence of detectable thyroid-
stimulating antibodies. In
one embodiment, the glucocorticoid is selected from the group comprising
dexamethasone,
prednisone, hydrocortisone, fluticasone, or cortisone. In one preferred
embodiment, the cultured
cells are selected from the group consisting of RDluc and CHORluc cells. In
another embodiment,
the observing is conducted using a luminometer. In further embodiments, the
cAMP concentration
is determined by the luciferin-luciferase system. In yet another embodiment,
the methods further
comprise a Growth Medium, while in other embodiments, the methods further
comprise a
Stimulation Medium. In some particularly preferred embodiments, the cultured
cells are exposed to
the Growth Medium prior to exposure of the test sample. In still further
embodiments, the cultured
cells are exposed to Stimulation Medium containing the test sample. In other
particularly preferred
embodiments, the Stimulation Medium comprises polyethylene glycol.
The present disclosure also provides methods for determining the presence of
thyroid-
stimulating autoantibodies in a test sample, comprising: a) providing; i) a
test sample suspected of
containing thyroid-stimulating autoantibodies, ii) cultured cells comprising a
glucocorticoid,
wherein the cells are selected from the group comprising RD-Rluc or CHO-Rluc
cells contained
within a testing means, wherein the cells express a chimeric TSH receptor, and
iii) polyethylene
glycol; b) exposing the test sample to the cultured cells and the polyethylene
glycol under conditions
such that thyroid stimulating antibodies are detectable using a luciferin-
luciferase system; and c)
observing for the presence of detectable thyroid-stimulating antibodies,
wherein observing is
conducted using a luminometer. In one embodiment, the glucocorticoid is
selected from the group
comprising dexamethasone, prednisone, hydrocortisone, fluticasone, or
cortisone. In further
embodiments, the cAMP concentration is determined by the luciferin-luciferase
system. In some
embodiments, the methods further comprise a Growth Medium, while in other
embodiments the
methods further comprise a Stimulation Medium. In some particularly preferred
embodiments, the
cultured cells are exposed to the Growth Medium prior to exposure of the test
sample. In still other
embodiments, the cultured cells are exposed to the Stimulation Medium
containing the test sample.
In yet other preferred embodiments, the Stimulation Medium comprises
polyethylene glycol.
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The present disclosure also provides methods for determining the presence of
thyroid-
stimulating autoantibodies in a test sample, comprising: a) providing; i) a
test sample suspected of
containing thyroid-stimulating autoantibodies, ii) cultured cells comprising a
glucocorticoid,
wherein the cells are selected from the group comprising RD-Rluc or CHO-Rluc
cells contained
within a testing means, wherein the cells express a chimeric TSH receptor,
iii) Growth Medium, and
iv) Stimulation Medium, wherein the Stimulation Medium comprises polyethylene
glycol; b)
exposing the cultured cells to Growth Medium to produce grown cells; c)
exposing the test sample
to the grown cells and Stimulation Medium under conditions such that thyroid-
stimulating
antibodies are detectable using the luciferin-luciferase system; and d)
observing for the presence of
detectable thyroid-stimulating antibodies, wherein said observing is conducted
using a luminometer.
In one embodiment, the glucocorticoid is selected from the group comprising
dexamethasone,
prednisone, hydrocortisone, fluticasone, or cortisone. In further embodiments,
the cAMP
concentration is determined by the luciferase-luciferin system.
Various embodiments of the claimed invention relate to a method for detecting
thyrotropin
stimulating hormone autoantibodies, comprising: a) providing; i) a non-starved
cell line comprising
a stably transfected vector encoding a chimeric thyrotropin stimulating
hormone (TSH) receptor and
a luciferase gene, wherein the chimeric TSH receptor comprises a human TSH
receptor amino acid
sequence wherein amino acid residues 262-335 of the human TSH receptor amino
acid sequence are
substituted with a corresponding portion of a rat chorionic gonadotrophin
receptor; ii) a serum
sample derived from a patient suspected of having Graves' disease; and iii) a
cell culture medium; b)
contacting the serum sample with the cell line and the medium for at least
sixteen hours
such that the luciferase gene emits a detectable signal.
Various embodiments of the claimed invention relate to a method for detecting
thyrotropin
stimulating hormone autoantibodies, comprising: a) providing: i) a test sample
suspected of
containing thyroid-stimulating autoantibodies; ii) non-starved cultured cells
contained within a
testing means, wherein said cells express a chimeric thryotropin stimulating
hormone (TSH)
receptor and a luciferase, wherein the chimeric TSH receptor comprises a human
TSH receptor
amino acid sequence wherein amino acid residues 262-335 of the human TSH
receptor amino acid
sequence are substituted with a corresponding portion of a rat chorionic
gonadotrophin receptor, and
iii) polyethylene glycol; b) exposing said test sample to said cultured cells
and said polyethylene
glycol for at least sixteen hours such that said thyroid-stimulating
antibodies are detectable using
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said luciferin-luciferase system; and c) observing for the presence of
detectable thyroid-stimulating
antibodies.
Various embodiments of the claimed invention relate to a kit comprising a cell
line
expressing a chimeric thyrotropin stimulating hormone (TSH) receptor, wherein
the chimeric TSH
receptor comprises a human TSH receptor amino acid sequence, wherein amino
acid residues 262-
335 of the human TSH receptor amino acid sequence are substituted with a
corresponding portion of
a rat chorionic gonadotrophin receptor, and a luciferin-luciferase system
capable of detecting serum
TSH autoantibodies.
Brief Description Of The Figures
Figure 1 provides results for serial 3-fold dilutions of three Graves' disease
IgG samples (from
untreated Graves' patients), in assays utilizing Stimulation Medium containing
6% PEG-8000.
Figure 2 provides a comparison of CHO-Rluc luciferase results with the FRTL-5
cAMP results
for IgGs from 35 untreated Graves' patients.
Figure 3 provides a comparison of CHO-Rluc luciferase results with CHO-R cAMP
results for
IgGs from 35 untreated Graves' patients.
Figure 4 provides a comparison of CHO-R cAMP results with FRTL-5 cAMP results
for IgGs
from 35 untreated Graves' patients.
Figure 5 shows the linearity of the response to bTSH of the CHO-Rluc cells.
Figure 6 shows the results for a group of samples with known TSI results using
FRTL-5 cells (10
1 samples of LCA TSI specimens).
Figure 7 shows the results for a group of normal samples (10 I of AML
"normal" specimens).
Figure 8 shows one embodiment of a DNA sequence for a chimeric hTSH/mLH (Mc4)
receptor
comprising 2,324 base pairs and encoding 730 amino acids. The underlined
letters are the human TSHR
sequence. The letters in italics are the rat LHR sequence. "* "(I) in the rat
LHR sequence is a G in the
wild type sequence. This G to T mutation resulted in an amino acid change from
Arginine to Serine.
Figure 9 shows one embodiment of a 236 nucleotide glycoprotein alpha subunit
promoter
comprising a cyclic AMP (cAMP) regulatory element (CRE) (AF401991) sequence
alignment with a
GPH promoter amplified by PCR from HEK cells. Shaded areas indicate homology.
Non-highlighted
areas designate the flanking region of the promoter in the plasmid.
Figure 10 presents exemplary data showing the response of the CHO-RMc4, RD-
RMc4 and
CHO-RLuc cell lines to negative and positive TSI-containing sera.
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Figure 10A: Luciferase assay on CHO-RLuc and CHO-RMc4 cell lines induced
with TSI negative and positive sera.
Figure 10B: The ratio of S/N derived from the luciferase assay on CHO-RLuc
and CHO-RMc4 cell lines induced with TSI negative and positive sera.
Figure 10C: Luciferase assay on CHO-RLuc and RD-RMc4 cell lines induced
with TSI negative and positive sera.
Figure 10D: The ratio of S/N derived from the luciferase assay on CHO-RLuc
and RD-RMc4 cell lines induced with TSI negative and positive sera.
Figure 10E: The ratio of S/N derived from the luciferase assay on CHO-RLuc,
CHO-RMc4 and RD-RIVIc4 cell lines induced with TSI negative and positive sera.
Figure 11 presents exemplary data showing signal-to-noise (S/N) ratios for RD-
RMc4 and CHO-RLuc cell lines in response to a serum dilution profile.
Figure 11A: The S/N ratio from the luciferase assay on CHO-RLuc and RD-
RMc4 cell lines induced with same dilutions of the TSI positive serum.
Figure 11B: The S/N ratio from the luciferase assay on CHO-RMc4 cell line
induced with dilution of the TSI positive serum.
Figure 11C: The S/N ratio from the luciferase assay on CHO-RMc4 cell line
induced with higher dilutions of the TSI positive
Figure 12 presents exemplary data comparing TSH sensitivity between a CHO-
-- RMc4 cell line and a CHO-RLuc cell line.
Figure 13 presents exemplary data presenting the distribution of signal-to-
noise
ratios from human sera using CHO-RMc4 and CHO-RLuc cell lines.
Figure 14 presents exemplary data showing the relative sensitivity of the CHO-
RMc4, RD-RMc4 and CHO-RLuc cell lines to clinical patient serum samples.
Figure 15 presents exemplary amino acid sequences: lutenizing hormone
receptor:
Figure 15A: Callithrix jacchus (white-tufted-ear marmoset) CAJ57370
Figure 15B: Cotumix japonica (Japanese quail) AAB32614
Figure 15C: Gallus gallus (chicken) NP_990267
Figure 15D: Mus musculus (mouse) AAB24402
Figure 15E: Bos taurus (cow) NP_776806
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Figure 16 illustrates a representative arrangement of TSI samples in a testing
plate.
Figure 17 present exemplary Relative Light Unit (RLU) data showing that the
alternative glucocorticoids fluticasone, prednisone, hydrocortisone and
cortisone provide
equal signal intensities of the CHO-RMc4 assay when compared to 40 jtM
dexamethasone.
Figure 18 present exemplary Serum Reference Unit percentages (SSR%) data
showing that the alternative glucocorticoids fluticasone, prednisone,
hydrocortisone and
cortisone provide an improved CHO-RMc4 assay.
Definitions
The terms "sample" and "specimen" in the present specification and claims are
used in their broadest sense. On the one hand, they are meant to include a
specimen or
culture. On the other hand, they are meant to include both biological and
environmental
samples. These terms encompass all types of samples obtained from humans and
other
animals, including but not limited to, body fluids (e.g., blood), as well as
solid tissue.
Biological samples may be animal, including human, fluid or tissue, food
products and ingredients such as dairy items, vegetables, meat and meat by-
products, and
waste. These examples are not to be construed as limiting the sample types
applicable to
the present invention.
As used herein, the term "kit" is used in reference to a combination of
reagents
and other materials.
As used herein, the term "antibody" is used in reference to any immunoglobulin
molecule that reacts with a specific antigen. It is intended that the term
encompass any
immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) obtained from any source
(e.g.,
humans, rodents, non-human primates, caprines, bovines, equines, ovines,
etc.).
As used herein, the term "antigen" is used in reference to any substance that
is
capable of reacting with an antibody. It is intended that this term encompass
any antigen
and "immunogen" (i.e., a substance which induces the formation of antibodies).
Thus, in
an immunogenic reaction, antibodies are produced in response to the presence
of an
antigen (immunogen) or portion of an antigen.
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As used herein, the terms "antigen fragment" and "portion of an antigen" are
used
in reference to a portion of an antigen. Antigen fragments or portions may
occur in
various sizes, ranging from a small percentage of the entire antigen to a
large percentage,
but not 100% of the antigen. However, in situations where at least a portion
of an antigen
is specified, it is contemplated that the entire antigen may be present. It is
contemplated
that antigen fragments or portions, may, but are not required to comprise an
"epitope"
recognized by an antibody. Antigen fragments or portions also may or may not
be
immunogenic.
As used herein, the term "autoantibodies" refers to antibodies that are
capable of
reacting against an antigenic constituent of an individual's own tissue or
cells (e.g., the
antibodies recognize and bind to "self' antigens).
As used herein, the term "immunoassay" is used in reference to any method in
which antibodies are used in the detection of an antigen. It is contemplated
that a range of
immunoassay formats be encompassed by this definition, including but not
limited to,
direct immunoassays, indirect immunoassays, and "sandwich" immunoassays."
However,
it is not intended that the present invention be limited to any particular
format. It is
contemplated that other formats, including radioimmunoassays (RIA),
immunofluorescent assays (IFA), and other assay formats, including, but not
limited to,
variations on the ELISA, RIA and/or IFA methods will be useful in the method
of the
present invention.
As used herein, the term "capture antibody" refers to an antibody that is used
to
bind an antigen and thereby permit the recognition of the antigen by a
subsequently
applied antibody. For example, the capture antibody may be bound to a
microtiter well
and serve to bind an antigen of interest present in a sample added to the
well. Another
antibody (termed the "primary antibody") is then used to bind to the antigen-
antibody
complex, in effect to form a "sandwich" comprised of antibody-antigen-antibody
complex. Detection of this complex can be performed by several methods. The
priniary
antibody may be prepared with a label such as biotin, an enzyme, a fluorescent
marker, or
radioactivity, and may be detected directly using this label. Alternatively, a
labelled
"secondary antibody" or "reporter antibody" which recognizes the primary
antibody may
be added, forming a complex comprised of an antibody-antigen-antibody-antibody
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complex. Again, appropriate reporter reagents are then added to detect the
labelled
antibody. Any number of additional antibodies may be added as desired. These
antibodies
may also be labelled with a marker, including, but not limited to an enzyme,
fluorescent
marker, or radioactivity.
As used herein, the term "reporter reagent" or "reporter molecule" is used in
reference to compounds which are capable of detecting the presence of antibody
bound to
antigen. For example, a reporter reagent may be a colorimetric substance
attached to an
enzymatic substrate. Upon binding of antibody and antigen, the enzyme acts on
its
substrate and causes the production of a color. Other reporter reagents
include, but are not
limited to, fluorogenic and radioactive compounds or molecules. This
definition also
encompasses the use of biotin and avidin-based compounds (e.g., including, but
not
limited to, neutravidin and streptavidin) as part of the detection system. In
one
embodiment of the present invention, biotinylated antibodies may be used in
the present
invention in conjunction with avidin-coated solid support.
As used herein the term "signal" is used in reference to an indicator that a
reaction
has occurred, for example, binding of antibody to antigen. It is contemplated
that signals
in the form of radioactivity, fluorogenic reactions, luminscent and enzymatic
reactions
will be used with the present invention. The signal may be assessed
quantitatively as well
as qualitatively.
As used herein the term "signal intensity" refers to magnitude of the signal
strength wherein the intensity correlates with the amount of reaction
substrate. For
example, a luciferin-luciferase system generates a signal intensity that
correlates with the
amount of cAMP generated by thyrotropin stimulating hormone receptor
autoantibodies.
As used herein, the term "luciferin-luciferase system" refers to any process
or
method that allows the contact of luciferin and luciferase in the presence of
a substrate
(i.e., for example, cAMP) under conditions such that the resulting luminesence
may be
detected. Such a system may be comprised within a transfected host cell
encoded by a
vector, or provided in separate kit containers whereby the contents may be
mixed
together.
As used herein, the term "solid support" is used in reference to any solid
material
to which reagents such as antibodies, antigens, and other compounds may be
attached.
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For example, in the ELISA method, the wells of microtiter plates often provide
solid
supports. Other examples of solid supports include microscope slides,
coverslips, beads,
particles, cell culture flasks, as well as many other items.
As used herein, the term "cell staining" is used in reference to methods used
to
label or stain cells to enhance their visualization. This staining or
labelling may be
achieved through the use of various compounds, including but not limited to,
fluorochromes, enzymes, gold, and iodine. It is contemplated that the
definition
encompasses such methods as "in situ chromogenic assays," in which a test
(i.e., an
assay) is conducted on a sample in situ. It is also contemplated that the in
situ
chrdmogenic assay will involve the use of an immunoassay (i.e., an ELISA).
As used herein, the term "Growth Medium" refers to a culture medium formulated
to contain various growth factors including, but not limited to, vitamins,
amino acids, co-
factors, and any other appropriate nutrients to enhance growth and replication
of cells in
culture.
As used herein, the term "Stimulation Medium" refers to a medium formulated to
be deficient in certain constituents (e.g., sodium chloride), in order to
enhance the
stimulation of by TSH and/or TSI, thereby increasing the resulting signal
(e.g., cAMP
and/or luciferase).
As used herein, the term "Starvation Medium" refers to a medium formulated to
be deficient in at least one growth factors included in the Growth Medium. In
preferred
embodiments, this medium contains only the salts and glucose necessary to
sustain cells
for a short period of time.
As used herein, the term "organism" and "microorganism," are used to refer to
any species or type of microorganism, including but not limited to viruses and
bacteria,
including rickettsia and chlamydia. Thus, the term encompasses, but is not
limited to
DNA and RNA viruses, as well as organisms within the orders Rickettsiales and
Chlamydiales.
As used herein, the term "culture," refers to any sample or specimen which is
suspected of containing one or more microorganisms. "Pure cultures" are
cultures in
which the organisms present are only of one strain of a particular genus and
species. This
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is in contrast to "mixed cultures," which are cultures in which more than one
genus
and/or species of microorganism are present.
As used herein, the term "cell type," refers to any cell, regardless of its
source or
characteristics.
As used herein, the term "cell line," refers to cells that are cultured in
vitro,
including primary cell lines, finite cell lines, continuous cell lines, and
transformed cell
lines.
As used herein, the terms "primary cell culture," and "primary culture," refer
to
cell cultures that have been directly obtained from animal or insect tissue.
These cultures
may be derived from adults as well as fetal tissue.
As used herein, the term "finite cell lines," refer to cell cultures that are
capable of
a limited number of population doublings prior to senescence.
As used herein, the term "continuous cell lines," refer to cell cultures that
have
undergone a "crisis" phase during which a population of cells in a primary or
finite cell
line apparently ceases to grow, but yet a population of cells emerges with the
general
characteristics of a reduced cell size, higher growth rate, higher cloning
efficiency,
increased tumorigenicity, and a variable chromosomal complement. These cells
often
result from spontaneous transformation in vitro. These cells have an
indefinite lifespan.
As used herein, the term "transformed cell lines," refers to cell cultures
that have
been transformed into continuous cell lines with the characteristics as
described above.
Transformed cell lines can be derived directly from tumor tissue and also by
in vitro
transformation of cells with whole virus (e.g., SV40 or EBV), or DNA fragments
derived
from a transforming virus using vector systems.
As used herein, the term "hybridomas," refers to cells produced by fusing two
cell
types together. Commonly used hybridomas include those created by the fusion
of
antibody-secreting B cells from an immunized animal, with a malignant myeloma
cell
line capable of indefinite growth in vitro. These cells are cloned and used to
prepare
monoclonal antibodies.
As used herein, the term "mixed cell culture," refers to a mixture of two
types of
cells. In some preferred embodiments, the cells are cell lines that are not
genetically
engineered, while in other preferred embodiments the cells are genetically
engineered cell
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lines. In some embodiments, the one or more of the cell types is "permissive"
(i.e., virus
is capable of replication and spread from cell to cell within the culture).
The present
invention encompasses any combination of cell types suitable for the
detection,
identification, and/or quantitation of viruses in samples, including mixed
cell cultures in
which all of the cell types used are not genetically engineered, mixtures in
which one or
more of the cell types are genetically engineered and the remaining cell types
are not
genetically engineered, and mixtures in which all of the cell types are
genetically
engineered.
As used herein, the term "suitable for the detection of intracellular
parasites,"
refers to cell cultures that can be successfully used to detect the presence
of an
intracellular parasite in a sample. In preferred embodiments, the cell
cultures are capable
of maintaining their susceptibility to infection and/or support replication of
the
intracellular parasite. It is not intended that the present invention be
limited to a particular
cell type or intracellular parasite.
As used herein, the term "susceptible to infection" refers to the ability of a
cell to
become infected with virus or another intracellular organism. Although it
encompasses
"permissive" infections, it is not intended that the term be so limited, as it
is intended that
the term encompass circumstances in which a cell is infected, but the organism
does not
necessarily replicate and/or spread from the infected cell to other cells. The
phrase "viral
proliferation," as used herein describes the spread or passage of infectious
virus from a
permissive cell type to additional cells of either a permissive or susceptible
character.
As used herein, the terms "monolayer," "monolayer culture," and "monolayer
cell
culture," refer to cells that have adhered to a substrate and grow as a layer
that is one cell
in thickness. Monolayers may be grown in various vessels including, but not
limited to,
flasks, tubes, coverslips (e.g., shell vials), roller bottles, etc. Cells may
also be grown
attached to microcarriers, including but not limited to beads.
As used herein, the term "suspension," and "suspension culture," refers to
cells
that survive and proliferate without being attached to a substrate. Suspension
cultures are
typically produced using hematopoietic cells, transformed cell lines, and
cells from
malignant tumors.
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As used herein, the terms "culture media," and "cell culture media," refers to
media that are suitable to support the growth of cells in vitro (i.e., cell
cultures). It is not
intended that the term be limited to any particular culture medium. For
example, it is
intended that the definition encompass outgrowth as well as maintenance media.
Indeed,
it is intended that the term encompass any culture medium suitable for the
growth of the
cell cultures of interest.
As used herein, the term "obligate intracellular parasite," (or "obligate
intracellular organism) refers to any organism which requires an intracellular
environment for its survival and/or replication. Obligate intracellular
parasites include
viruses, as well as many other organisms, including certain bacteria
including, but not
limited to, most members of the orders: i) Rickettsiales: for example,
Coxiella, Rickettsia
and Ehrlichia; and ii) Chlamydiales: for example, C. trachomatis, C. psittaci.
The term
"intracellular parasite," refers to any organism that may be found within the
cells of a
host animal, including but not limited to obligate intracellular parasites
briefly described
above. For example, intracellular parasites include organisms such as
Brucella, Listeria,
Mycobacterium (e.g., M. tuberculosis and M. leprae), and Plasmodium, as well
as
Rochalimea.
As used herein, the term "antimicrobial," is used in reference to any compound
which inhibits the growth of, or kills microorganisms. It is intended that the
term be used
in its broadest sense, and includes, but is not limited to compounds such as
antibiotics
which are produced naturally or synthetically. It is also intended that the
term includes
compounds and elements that are useful for inhibiting the growth of, or
killing
microorganisms.
As used herein, the terms "chromogenic compound," and "chromogenic
substrate," refer to any compound useful in detection systems by their light
absorption or
emission characteristics. The term is intended to encompass any enzymatic
cleavage
products, soluble, as well as insoluble, which are detectable either visually
or with optical
machinery. Included within the designation "chromogenic" are all enzymatic
substrates
which produce an end product which is detectable as a color change. This
includes, but is
not limited to any color, as used in the traditional sense of "colors," such
as indigo, blue,
red, yellow, green, orange, brown, etc., as well as fluorochromic or
fluorogenic
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compounds, which produce colors detectable with fluorescence (e.g., the yellow-
green of
fluorescein, the red of rhodamine, etc.). It is intended that such other
indicators as dyes
(e.g., pH) and luminogenic compounds be encompassed within this definition.
As used herein, the commonly used meaning of the terms "pH indicator," "redox
indicator," and "oxidation-reduction indicator," are intended. Thus, "pH
indicator,"
encompasses all compounds commonly used for detection of pH changes,
including, but
not limited to phenol red, neutral red, bromthymol blue, bromcresol purple,
bromcresol
green, bromchlorophenol blue, m-cresol purple, thym.ol blue, bromcresol
purple, xylenol
blue, methyl red, methyl orange, and cresol red. The terms "redox indicator,"
and
"oxidation-reduction indicator," encompasses all compounds commonly used for
detection of oxidation/reduction potentials (i.e., "eH") including, but not
limited to
various types or forms of tetrazolium, resazurin, and methylene blue.
As used herein, the term "inoculating suspension," or "inoculant," is used in
reference to a suspension which may be inoculated with organisms to be tested.
It is not
intended that the term "inoculating suspension," be limited to a particular
fluid or liquid
substance. For example, inoculating suspensions may be comprised of water,
saline, or an
aqueous solution. It is also contemplated that an inoculating suspension may
include a
component to which water, saline or any aqueous material is added. It is
contemplated in
one embodiment, that the component comprises at least one component useful for
the
intended microorganism. It is not intended that the present invention be
limited to a
particular component.
As used herein, the term "primary isolation," refers to the process of
culturing
organisms directly from a sample. As used herein, the term "isolation," refers
to any
cultivation of organisms, whether it be primary isolation or any subsequent
cultivation,
including "passage," or "transfer," of stock cultures of organisms for
maintenance and/or
use.
As used herein, the term "presumptive diagnosis," refers to a preliminary
diagnosis which gives some guidance to the treating physician as to the
etiologic
organism involved in the patient's disease. Presumptive diagnoses are often
based on
"presumptive identifications," which as used herein refer to the preliminary
identification
of a microorganism.
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As used herein, the term "definitive diagnosis," is used to refer to a final
diagnosis
in which the etiologic agent of the patient's disease has been identified. The
term
"definitive identification" is used in reference to the final identification
of an organism to
the genus and/or species level.
The term "recombinant DNA molecule," as used herein refers to a DNA molecule
which is comprised of segments of DNA joined together by means of molecular
biological techniques.
DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides
are reacted to make oligonucleotides in a manner such that the 5' phosphate of
one
mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in
one direction
via a phosphodiester linkage. Therefore, an end of an oligonucleotides is
referred to as
the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a
mononucleotide pentose
ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a
subsequent
mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if
internal to
a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a
linear or
circular DNA molecule, discrete elements are referred to as being "upstream"
or 5' of the
"downstream" or 3' elements. This terminology reflects the fact that
transcription
proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer
elements
which direct transcription of a linked gene are generally located 5' or
upstream of the
coding region (enhancer elements can exert their effect even when located 3'
of the
promoter element and the coding region). Transcription termination and
polyadenylation
signals are located 3' or downstream of the coding region.
The term "an oligonucleotide having a nucleotide sequence encoding a gene,"
refers to a DNA sequence comprising the coding region of a gene or, in other
words, the
DNA sequence which encodes a gene product. The coding region may be present in
either a cDNA or genomic DNA form. Suitable control elements such as
enhancers,
promoters, splice junctions, polyadenylation signals, etc. may be placed in
close
proximity to the coding region of the gene if needed to permit proper
initiation of
transcription and/or correct processing of the primary RNA transcript.
Alternatively, the
coding region utilized in the vectors of the present invention may contain
endogenous
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enhancers and/or promoters, splice junctions, intervening sequences,
polyadenylation
signals, etc. or a combination of both endogenous and exogenous control
elements.
The term "transcription unit," as used herein refers to the segment of DNA
between the sites of initiation and termination of transcription and the
regulatory
elements necessary for the efficient initiation and termination. For example,
a segment of
DNA comprising an enhancer/promoter, a coding region, and a termination and
polyadenylation sequence comprises a transcription unit.
The term "regulatory element," as used herein refers to a genetic element
which
controls some aspect of the expression of nucleic acid sequences. For example,
a
promoter is a regulatory element which facilitates the initiation of
transcription of an
operably linked coding region. Other regulatory elements are splicing signals,
polyadenylation signals, termination signals, etc. (defined infra).
The terms "reporter gene construct," or "reporter gene vector," as used herein
refers to a recombinant DNA molecule containing a sequence encoding the
product of a
reporter gene and appropriate nucleic acid sequences necessary for the
expression of the
operably linked coding sequence in a particular host organism. Eukaryotic
cells are
known to utilize promoters, enhancers, and termination and polyadenylation
signals.
The term "reporter gene," refers to an oligonucleotide having a sequence
encoding
a gene product (typically an enzyme) which is easily and quantifiably assayed
when a
construct comprising the reporter gene sequence operably linked to a
heterologous
promoter and/or enhancer element is introduced into cells containing (or which
can be
made to contain) the factors necessary for the activation of the promoter
and/or enhancer
elements. Examples of reporter genes include but are not limited to bacterial
genes
encoding P-galactosidase (lacZ, the bacterial chloramphenicol
acetyltransferase (cat)
genes, firefly luciferase genes and genes encoding P-glucuronidase (GUS).
Transcriptional control signals in eukaryotes comprise "promoter" and
"enhancer"
elements. Promoters and enhancers consist of short arrays of DNA sequences
that interact
specifically with cellular proteins involved in transcription (Maniatis, et
al., Science
236:1237 (1987)). Promoter and enhancer elements have been isolated from a
variety of
eukaryotic sources including genes in yeast, insect and mammalian cells and
viruses
(analogous control elements, i.e., promoters, are also found in prokaryotes).
The selection
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of a particular promoter and enhancer depends onwhat cell type is to be used
to express
the protein of interest. Some eukaryotic promoters and enhancers have a broad
host range
while others are functional in a limited subset of cell types (for review see
Voss, et al.,
Trends Biochem. Sci., 11:287 (1986), and Maniatis, et al., supra (1987)). For
example,
the SV40 early gene enhancer is very active in a wide variety of cell types
from many
mammalian species and has been widely used for the expression of proteins in
mammalian cells (Dijkema, et al., EMBO J. 4:761 (1985)). Two other examples of
promoter/enhancer elements active in a broad range of mammalian cell types are
those
from the human elongation factor la gene (Uetsuki et al., J. Biol. Chem.,
264:5791
(1989); Kim et al., Gene 91:217 (1990); and Mizushima and Nagata, Nuc. Acids.
Res.,
18:5322 (1990)) and the long terminal repeats of the Rous sarcoma virus
(Gorman et aL,
Proc. Natl. Acad. Sci. USA 79:6777 (1982)), and the human cytomegalovirus
(Boshart et
aL, Cell 41:521 (1985)).
The term "promoter/enhancer," denotes a segment of DNA which contains
sequences capable of providing both promoter and enhancer functions (for
example, the
long terminal repeats of retroviruses contain both promoter and enhancer
functions). The
enhancer/promoter may be "endogenous," or "exogenous," or "heterologous." An
endogenous enhancer/promoter is one which is naturally linked with a given
gene in the
genome. An exogenous (heterologous) enhancer/promoter is one which is placed
in
juxtaposition to a gene by means of genetic manipulation (i.e., molecular
biological
techniques).
The presence of "splicing signals," on an expression vector often results in
higher
levels of expression of the recombinant transcript. Splicing signals mediate
the removal
of introns from the primary RNA transcript and consist of a splice donor and
acceptor site
(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring
Harbor
Laboratory Press, New York (1989), pp. 16.7-16.8). A commonly used splice
donor and
acceptor site is the splice junction from the 16S RNA of SV40.
Efficient expression of recombinant DNA sequences in eukaryotic cells requires
signals directing the efficient termination and polyadenylation of the
resulting transcript.
Transcription termination signals are generally found downstream of the
polyadenylation
signal and are a few hundred nucleotides in length. The term "poly A site," or
"poly A
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sequence," as used herein denotes a DNA sequence which directs both the
termination
and polyadenylation of the nascent RNA transcript. Efficient polyadenylation
of the
recombinant transcript is desirable as transcripts lacking a poly A tail are
unstable and are
rapidly degraded. The poly A signal utilized in an expression vector may be
"heterologous" or "endogenous." An endogenous poly A signal is one that is
found
naturally at the 3' end of the coding region of a given gene in the genome. A
heterologous
poly A signal is one which is isolated from one gene and placed 3' of another
gene. A
commonly used heterologous poly A signal is the SV40 poly A signal. The SV40
poly A
signal is contained on a 237 bp BamHI/BcII restriction fragment and directs
both
termination and polyadenylation (Sambrook, supra, at 16.6-16.7). This 237 bp
fragment
is contained within a 671 bp BarnHI/PstI restriction fragment.
The term "genetically engineered cell line," refers to a cell line that
contains
heterologous DNA introduced into the cell line by means of molecular
biological
techniques (i.e., recombinant DNA technology).
The term "vector" as used herein, refers to a nucleotide sequence comprising
at
least a promoter and a gene of interest. Such a gene of interest may encode an
amino
acid sequence for the purpose of expressing the amino acid sequence (i.e., for
example, a
TSH receptor amino acid sequence). A vector has the capability of becoming
integrated
into foreign DNA to form a stable transfected cell.
The term "stable transfection," or "stably transfected," refers to the
introduction
and integration of foreign DNA into the genome of the transfected cell.
The term "stable transfectant," refers to a cell which has stably integrated
foreign
DNA into the genomic DNA.
The term "stable transfection" (or "stably transfected"), refers to the
introduction
and integration of foreign DNA into the genome of the transfected cell. The
term "stable
transfectant," refers to a cell which has stably integrated foreign DNA into
the genomic
DNA.
The term "RDluc" refers to an RD cell line having been stably transfected with
a
luciferase gene. Further, RD-Rluc refers to a RD cell line having been stably
transfected
with a luciferase gene and that displays an exogenous receptor (i.e., for
example, a TSH
receptor including but not limited to, a Mc4 receptor).
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The term "CHOluc" refers to a CHO cell line having been stably transfected
with
a luciferase gene. Further, CHO-Rluc refers to a CHO cell line having been
stably
transfected with a luciferase gene and that displays an exogenous receptor
(i.e., for
example, a TSH receptor including but not limited to, a wild-type receptor).
Alternatively, CHO-RMc4luc refers to a CHO cell line having been stably
trasnfected
with a luciferase gene that displays a chimeric receptor. (i.e., for example,
a TSH
receptor that comprises amino acid sequences derived from a rat chorionic
gonadotrophin
receptor).
The term "selectable marker," as used herein refers to the use of a gene which
encodes an enzymatic activity that confers resistance to an antibiotic or drug
upon the cell
in which the selectable marker is expressed. Selectable markers may be
"dominant"; a
dominant selectable marker encodes an enzymatic activity which can be detected
in any
mammalian cell line. Examples of dominant selectable markers include the
bacterial
aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene)
which
confers resistance to the drug G418 in mammalian cells, the bacterial
hygromycin G
phosphotransferase (hyg) gene which confers resistance to the antibiotic
hygromycin and
the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred
to as the gpt
gene) which confers the ability to grow in the presence of mycophenolic acid.
Other
selectable markers are not dominant in that their use must be in conjunction
with a cell
line that lacks the relevant enzyme activity. Examples of non-dominant
selectable
markers include the thymidine kinase (tk) gene which is used in conjunction
with tk cell
lines, the CAD gene which is used in conjunction with CAD-deficient cells and
the
mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which is
used
in conjunction with hprt cell lines. A review of the use of selectable markers
in
mammalian cell lines is provided in Sambrook et al., supra at pp.16.9-16.15.
The terms "nucleic acid molecule encoding," "DNA sequence encoding," and
"DNA encoding," refer to the order or sequence of deoxyribonucleotides along a
strand
of deoxyribonucleic acid. The order of these deoxyribonucleotides determines
the order
of amino acids along the polypeptide (protein) chain. The DNA sequence thus
codes for
the amino acid sequence.
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The terms "confluent" or "confluency" as used herein in reference to an
adherent
cell line define a condition wherein cells throughout a culture are in contact
with each
other creating what appears to be a continuous sheet or "monolayer" of cells.
The terms "cytopathic effect" or "CPE" as used herein describe changes in
cellular structure (i.e., a pathologic effect) resulting from external agents
such viruses.
Common cytopathic effects include cell destruction, syncytia (i.e., fused
giant cells)
formation, cell rounding vacuole formation, and formation of inclusion bodies.
CPE
results from actions of a virus on permissive cells that negatively affect the
ability of the
permissive cellular host to preform its required functions to remain viable.
In in vitro cell
culture systems, CPE is evident when cells, as part of a confluent monolayer,
show
regions of non-confluence after contact with a specimen that contains a virus.
The
observed microscopic effect is generally focal in nature and the foci is
initiated by a
single virion. However, depending upon viral load in the sample, CPE may be
observed
throughout the monolayer after a sufficient period of incubation. Cells
demonstrating
viral induced CPE usually change morphology to a rounded shape, and over a
prolonged
period of time can die and be released form their anchorage points in the
monolayer.
When many cells reach the point of focal destruction, the area is called a
viral plaque,
which appears as a hole in the monolayer. Cytopathic effects are readily
discernable and
distinguishable by those skilled in the art.
The abbreviation "ONPG," represents o-Nitropheny1-13-D-Galactopyranoside.
ONPG is a substrate for the enzyme P-galactosidase (p-gal). The reaction
between ONPG
and (P-gal produces a yellow product which can be quantified
spectrophotometrically at
405 nm.
The abbreviation "X-gal," represents the chemical compound 5-bromo-4-chloro-
3-indoly1-(3-D-galactopyranoside, a substrate for the enzyme p-galactosidase.
The
reaction between X-gal and P-galactosidase results in the formation of a blue
precipitate
which is visually discernable.
The term "hybriwix," represents a product of Diagnostic Hybrids, Inc., Athens,
Ohio which allows for quantification of certain viral DNA in an infected
monolayer of
cells by DNA hybridization. "DNA hybridization" is the annealing of two
complementary
DNA molecules whose base sequences match according to the rules of base
pairing.
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DNA hybridization is used to identify or quantify an unknown or "target" DNA
by
hybridization to a known DNA or "probe." The probe is typically labeled with a
reporter
molecule such as 1251, a radioisotope which can be detected and quantified
with a gamma
counter.
The phrase "plaque reduction assay," or "PRA," as used herein describes a
standard method used to determine efficacy of anti-viral drugs by enumerating
a decrease
in plaque formation in a cell monolayer exposed to a drug. A "plaque" is a
defined area
of "CPE." It is usually the result of infection of the cell monolayer with a
single
infectious virus which then replicates and spreads to adjacent cells of the
monolayer. A
plaque may also be referred to as a "focus of viral infection."
The term "permissive" as used herein describes the sequence of interactive
events
between a virus and its putative host cell. The process begins with viral
adsorption to the
host cell surface and ends with release of infectious virions. A cell is
"permissive" if it
readily permits the spread of virus to other cells. Many methods are available
for the
determination of the permissiveness of a given cell line, including but not
limited to,
plaque reduction assays, comparisons of the production and/or quantitation of
viral
proteins based on results obtained from gel electrophoresis, relative
comparisons using
hybridization analysis to analyze DNA or RNA content, etc.
The term "susceptible," as used herein describes the extent that a permissive
or
non-permissive host cell can adsorb and be penetrated by a virus. A cell line
may be
susceptible without being permissive in that it can be penetrated but not
release virions. A
permissive cell line however must be susceptible.
The phrase "seed on," as used herein describes the act of transferring an
aqueous
solution of suspended ceils into a vessel containing cells adhered to a
surface, after which
the vessel is stored for a sufficient period of time to allow the suspended
cells or "seeds"
to settle out by gravity and attach in a relatively uniform manner to the
adhered cells and
become integrated into the final cell monolayer as a mixture. A "mixed cell
monolayer,"
results from the "seed on" process.
The phrase "seed in," as used herein describes the mixing of two or more
aqueous
solutions of suspended tissue culture cells, each cell suspension having
different cellular
properties, and transfer of such mixture of ceils into a vessel which is
stored for a
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sufficient period of time to allow the suspended cells to settle out by
gravity and attach in
a relatively uniform manner such that the distribution of any single cell type
is indicative
of the relative ratio of the cells in the original mixture.
The term "starts," as used herein refers to the reporter cells which represent
a
primary infection of virus. The virus infects a reporter cell (a genetically
engineered cell)
and induces the expression of the reporter gene. A reporter cell can be
nonpermissive (i.e.
permissiveness of the reporter cells is not required) and still produce
starts.
The term "chimeric" as used herein, refers to any nucleic and/or amino acid
sequence containing portions from two or more different species. A protein may
be
chimeric if the primary amino acid sequence contains portions from two or more
different
species (i.e., for example, an hTSH/rLH-R or RMc4). A protein may also be
chimeric if
the primary amino acids sequence contains portions from two or more different
proteins,
whether from the same species or different species. A protein may also be
chimeric if the
quaternary amino acid structure contains proteins from two or more different
species.
Further, a nucleic acid may be chimeric if the primary nucleotide sequence
contains
portions from two or more different species. A nucleic acid may also be
chimeric if the
primary nucleotide sequence contains portions from two or more different
proteins,
whether from the same species or different species.
The term, "glucocorticoid" as used herein, refers to any compound any
corticosteroid that increases gluconeogenesis, raising the concentration of
liver glycogen
and blood glucose; the group includes, but is not limited to, dexamethasone,
prednisone,
hydrocortisone, fluticasone, cortisol, cortisone, or corticosterone.
Detailed Description
The present invention provides methods and compositions useful in the
diagnosis
of autoimmune diseases. In particular, the present invention provides methods
and
compositions for use in the diagnosis and management of Graves' disease. For
example,
one composition comprises a chimeric thyroid stimulating hormone receptor
having
improved sensitivity and specificty for circulating thyroid stimulating
immunoglobulin.
Assays using such chimeric receptors can be optimized in the presence of a
glucocorticoid.
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In addition, the present invention provides methods and compositions for
monitoring the immune status and responses of individuals. In particular, the
present
invention finds use in montoring the immune responses of vaccine recipients.
The
present invention further provides methods and compositions for accelerating
and
enhancing the attachment of viruses to cell surface receptors, providing
increased
sensitivity in assays to detect and quantitate viruses in samples.
I. Graves' Disease
Typically, the clinical picture of Graves' disease in young adults is very
easily
recognized. The patients are more commonly female than male, and report
symptoms
including, but not limited to, sweating, palpitations, nervousness,
irritability, insomnia,
tremor, frequent stools, and weight loss in spite of a good appetite. Physical
examination
usually shows mild proptosis, stare, lid lag, a smooth, diffuse, non-tender
goiter,
tachycardia (especially after exercise) with loud heart sounds, and often a
systolic
murmur or left sternal border scratch, tremor, onycholysis, and palmar
erythema; often, a
bruit is heard over the thyroid, and a cervical hum is almost always present.
In patients
with these symptoms, Graves' disease is readily recognized, and can be
confirmed with
laboratory tests (See, Federman, Thyroid, in Dale and Federman (eds.),
Scientific
American Medicine, 3:1-6, Scientific American, New York, NY, (1997).
Although the signs and symptoms described above can be troublesome, other
symptoms of the disease can be more dangerous. One of the most disturbing
symptoms is
severe exophthalmos, accompanied by ophthalmoplegia, follicular
conjunctivitis,
chemosis, and loss of vision. Additional symptoms include, but are not limited
to,
dermopathy, pretibial myxedema, clubbing, and in the most severe cases,
acropachy.
These signs and symptoms are indicative of a representative autoimmune
etiology of
Graves' disease.
Despite the typical clinical picture of Graves' disease, methods are needed to
confirm the diagnosis, as well as provide prognostic indicators for management
and
treatment. In addition, in cases where the cause of hyperthyroidism is
unclear, diagnostic
test methods must be utilized to determine the etiology. Although in vivo
methods such
as radioactive-iodine uptake (RAIU) may be used in the diagnosis and
monitoring of
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patients with Graves' disease (See e.g.,. Baldet et al., Acta
Endocrinol.(Copenh) 116:7-12
(1987)), there are two basic groups of in vitro assay systems developed for
this purpose.
One is dependent upon the measurement of some index of thyroid stimulation
(e.g.,
cAMP generation) and the other assesses the ability of thyroid-stimulating
autoantibodies
(TSAb) to inhibit the binding of radiolabelled thyroid stimulating hormone
(TSH) to its
receptor. These methods include bioassays and in vitro assays for TSAb.
However, no
widespread application of methods to measure the thyroid-stimulating
immunoglobulin
(TSI or TSAb) in Graves' disease diagnosis has been reported. (See e.g.,
Rapoport et aL,
J. Clin. Endocrinol.Metabol., 58:332-338 (1984)). In addition, it was
recognized that in
the sera of Graves' disease patients there is a heterogenous population of
immunoglobulin
G (IgG) molecules that recognize the thyroid hormone receptor (See e.g.,
Yokoyama et
aL, J. Clin. Endocrinol. Metabol., 64:215-218 1987)). Further, the recognition
that TSH-
binding inhibition assays do not necessarily reflect a thyroid-stimulating
activity
contributed to confusion in attempts to reach agreement on the clinical
application of
such assays (See e.g., McKenzie and Zakarija, J. Clin. Endocrinol. Metabol.,
69:1093-
1096 (1989)). Limitations in terms of sensitivity and specificity were also
problematic.
Indeed, problems associated with available assay systems resulted in arguments
that the
measurement of thyroid peroxidase antibodies is a sufficiently sensitive
marker for
underlying thyroid autoimmunity (See, Botero and Brown, supra).
As indicated by Rapoport et al., the available assays that could be performed
easily, in a standardized manner, and for large numbers of samples had
significant
limitations in terms of sensitivity and/or specificity, making these tests
unreliable for
clinical use. These problems apply primarily to assays that measure the
ability of TSI to
inhibit the binding of radiolabelled TSH to human thyroid plasma membranes
(i.e., the
assays do not measure TSI activity per se). Also, not all of the anti-TSH
receptor
antibodies are stimulatory. Rapoport et al. further indicate that assays using
TSI
stimulation of adenylate cyclase activity in human thyroid plasma membranes
are
seriously lacking in sensitivity. Some assays are unpractical for general
clinical use,
including those that rely upon the use of fresh human thyroid tissue, involve
extremely
difficult techniques with limited sample capacity, and are very laborious
and/or
uneconomical (See e.g., Rapoport et al, supra). The development of assays
using cultured
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canine and porcine thyroid cells to measure the cAMP response to TSH were
later
adapted for use with human thyroid cells which offered potentially superior
results. In
addition to the requirement for fresh thyroid cells in some of these methods
(e.g., the
methods discussed by Rapoport et al.), many also required tedious and
timeconsuming
-- sample preparation prior to assaying the specimens. For example, some
protocols require
laborious and time-consuming dialysis methods and/or precipitation of
immunoglobulins
in the test sera with ammonium sulfate or polyethylene glycol (See e.g.,
Rapoport et al.,
supra; and Kasagi et al., J. Clin. Endocrinol. Metabol., 62:855-862 (1986)).
In view of the problems encountered with these assay systems, other methods
-- were investigated in an effort to develop an assay that is easy to perform,
reliable,
sensitive, and specific for Graves' disease autoantibodies. For example, the
use of
bioassays to measure cAMP production rely upon the use of cells of non-human
origin
grown in continuous culture or on human cells used as primary cultures or
frozen in
aliquots for use as needed. Problems with the use of human thyroid cells
include the
-- variability in responsiveness of surgically obtained thyroid tissue. Thus,
cells of
nonhuman origin gained popularity, including the rat thyroid cell line (FRTL-
5). This is a
non-transformed, differentiated cell line that has been well-studied and
characterized (See
e.g., Bidey et al., J. Endocrinol., 105:7-15 (1985); and Michelangeli et al.,
Clin.
Endocrinol.,40:645-652 (1994)). However, a number of disadvantages make these
cells
-- less than ideal for Graves' disease assays. For example, the cells are slow
growing and
have fastidious growth requirements which include the need for TSH.
Consequently, it is
necessary to deprive the cells of TSH for at least 5 days prior to assay in
order to achieve
a reasonable level of sensitivity.
Subsequent development of cells such as the JP09 cells (Chinese hamster ovary
-- cells transfected with a functional human TSH receptor) and other cell
lines which stably
express the human TSH receptor have greatly improved the assay systems
available for
the detection of Graves' disease autoantibodies. These cells have a TSH
receptor that is
comparable to that of native thyrocytes and possess a functional signal
transduction
system involving G-protein coupling, activation of adenylate cyclase and cAMP
-- generation in response to TSH and to thyroid-stimulating antibodies (TSAb)
(See e.g.,
Michelangeli et al., supra). These cells have been reported to be superior to
FRTL-5 cells
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as they provide similar diagnostic information, but are more sensitive, grow
faster, have
less fastidious growth requirements, and respond to unextracted sera, in
comparison with
FRTL-5 cells (Michelangeli et al., supra; see also, Kakinuma et al., J. Clin.
Endocrinol.
Metabol., 82:212902134 (1997)). In addition, these methods are more rapid and
reproducible, and perhaps more specific for detection of human autoantibodies
directed
against the human receptor. Further, the assays are easier and less cumbersome
to
perform than those using the FRTL-5 cell line (See e.g., Vitti etal., J. Clin.
Endocrinol.
Metabol., 76:499-503 (1993)). However, these assays rely upon the use of
radioactivity
(e.g., in radioimmunoassays) to detect and quantitate cAMP and are as a
result, still
cumbersome.
Diagnostic Assays For Graves Disease
Graves' disease is a thyroid disorder caused by an antibody-mediated auto-
immune reaction. In Graves' patients, the autoantibodies recognizing the TSHR
(TRAbs)
are heterogeneous, including mainly thyroid stimulating antibodies (TSAbs) and
thyroid
blocking antibodies (TBAbs.) TSAbs act as a TSH agonist causing
hyperthyroidism
while the TBAbs function as a TSH antagonist causing hypothyroidism. While
TSAb and
TBAb bind to different epitopes on the TSHR, TBAb binding can "neutralize" the
stimulating effect of TSAb. When the TSAb binds to the TSHR, it induces the
cAMP
signaling pathway, TBAb does not have this effect.
Currently, several bioassays are used to diagnose Graves' disease. The Kronus
Radio Receptor Assay (RRA) kit is used for determination of TRAbs and detects
both
TSAbs and TBAbs but cannot distinguish between the two. Diagnostic Hybrids
Inc.
(DHI) previously developed a Graves' diagnostic CHO-Luc cell line that detects
the
TRAbs in patient serum. This cell line co-expresses the wildtype TSH receptor
gene and
a firefly luciferase gene which is driven by the human glycoprotein alpha
subunit
promoter. This wild type TSHR has epitopes that bind TSAbs and TBAbs. Binding
of
TBAb to the receptor can modulate TSAbs' binding, resulting in lower
stimulation by the
TSAbs.
Thyroid-stimulating autoantibodies (TSAb) directed against the thyroid
stimulating hormone (TSH) receptor are capable of stimulating thyroid adenylyl
cyclase,
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the enzyme responsible for producing cyclic-adenosine monophosphate (cAMP).
These
autoantibodies have been used as diagnostic markers for detection and
identification of
patients suffering from Graves' disease, as these autoantibodies appear to be
responsible
for the hyperthyroidism seen in patients with this disease. However, as
discussed in more
detail below, the methods commonly used to detect and measure these TSAbs are
complex and time-consuming.
A. cAMP Detection
One method that measures TSAbs utilizes a rat thyroid cell line known as "FRTL-
5." This cell line, available from Interthyroid Research Foundation
(Baltimore, MD)
expresses receptors that cross-react with human TSAbs. In the presence of
TSAbs (i.e.,
for example, upon exposure of the cells to serum from a Graves' patient
containing these
antibodies), FRTL-5 cells are stimulated to produce cAMP. This cAMP is then
measured
in a portion of the lysed cells or the medium bathing the cells using a
radioimmunoassay
method. The FRTL-5 cells formed the basis for the first successful bioassay
for the
autoantibodies that are pathognomonic of Graves' disease. U.S. Patent No.
4,609,622
(herein incorporated by reference).
B. FRTL-5 Cell Assays And Starvation Medium
A typical assay using FRTL-5 cells performed as described by Vitti et al.
(Vitti et
al., J. Clin. Endocrinol. Metabol., 76:499 (1993)) involves seeding FRTL-5
cells in 96-
well plates (30,000 cells/well) in a special complete medium containing 6
hormones (i.e.,
for example, a 6H medium) in addition to the normal growth constituents used
in cell
culture medium. After 2-3 days incubation in a 5% CO2, humidified, 37 C
incubator (i.e.,
when the cells are confluent), the medium is changed to a "Starvation Medium,"
which is
deficient in TSH (thereby resulting in a 5H medium), wherein TSH is one of the
6
hormones in the 6H medium. The cells are then maintained for 4-5 days in the
incubator
with a medium change every 2-3 days. During this time the cells do not grow or
multiply.
Subsequently, the cells may be used in a diagnostic assay.
C. Radiolabel Assays And Stimulation Medium
Early diagnostic methods for Graves' disease were performed by removing the
Starvation Medium and adding a Simulation Medium comprising a special low
sodium
chloride, high sucrose buffer (HBSS NaC1 + 222 mM sucrose; the formula for
this buffer
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is: 0.0608 g/L KH2PO4, 0.144 g/L CaC12, 0.373 g/L KC1, 0.048 g/L MgSO4, 0.097
g/L
Na2PH04, 1.0 g/L D-glucose, 76 g/L (i.e., 222 mM) sucrose, 4.77 g/L HEPES, and
10
g/L BSA; pH 7.2 - 7.4) containing a phosphodiesterase inhibitor (e.g., 0.5 mM
methylisobutylxanthine; IBMX), to prevent this enzyme from breaking down cAMP.
Specially prepared samples of patient immunoglobulin (IgG), controls, and
standards are
added to the appropriate wells, usually in triplicate, and the plate is
incubated in a 5%
CO2, humidified, 37 C incubator for 2 hours. Following this incubation, 5-10
pi of the
medium are removed from each well and used in a radioimmunoassay system to
detect
the presence of cAMP. Typically this assay is run with about 6 standards in
duplicate,
with patient and controls also run in duplicate. The assay usually requires an
overnight
incubation with about an hour required the next day for the separation of
free,
radiolabelled cAMP from antibody-bound, radiolabelled cAMP.
As the use of radioactivity and long preparation times are negative aspects of
the
FRTL-5 assay, improved systems have been developed. One investigation involved
the
use of low salt conditions to increase the sensitivity of the assay system
(See, Kosugi et
al., Endocrinol., 125:410-417 (1989)). Additional improvements in the bioassay
involved
a strain of Chinese Hamster Ovary ("CHO") cells transfected with a human TSH
receptor
("CHO-R"; See, Vitti et al., supra). This cell line offered two major
improvements over
the FRTL assay. First, this method involves the use of human TSH receptors
instead of
rat TSH receptors which should provide greater specificity and perhaps
sensitivity for the
detection of TSAbs. Second, there is no requirement for the special 6H medium
and 5H
medium changes over a 6-8 day period, since the CHO-R cells grow well on a
standard
supplemented medium and can be used 1-3 days after seeding, depending on the
density
of the cell suspension used to inoculate the wells. In addition, comparative
studies with
FRTL-5 cells have shown that the CHO-R cells may be more accurate in detecting
Graves' TSAbs (See, Vitti et al.).
D. Luciferase Gene Assays Using CHO-Rluc Cell Lines
A further improvement was provided by the use of CHO-R cells designed to
readily assess the increased amounts of cAMP caused by TSI through the use of
a
reporter gene (i.e., for example, luciferase) (Evans et al., J. Clin.
Endocrinol. Metabol.,
84:374 (1999)). Thus, with the introduction of this engineered cell line
(i.e., CHO-Rluc),
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the complexity and dangers inherent in the use of radioactive compounds used
in the
.previously developed radioimmunoassay for cAMP detection and quantitation are
eliminated. With these cells, luciferase is measured simply by removing the
medium from
the cells, adding a lysis buffer, allowing 20-30 minutes for lysis to occur,
removing a
sample of the lysate, adding luciferase substrate and measuring light output
over a 15
second interval using a luminometer. However, as indicated in the Experimental
section
below, this method provides equivocal results and required further
improvement.
In one embodiment, the present invention contemplates methods that incorporate
the advantages of a CHO-Rluc protocol, while providing additional advantages
in terms
of reliability and reproducibility. Considerable development effort was
dedicated to the
development of methods of the present invention, including those that allow
the use of
CHO-Rluc cells in luminometric assays using TSH and immunoglobulins from
untreated
Graves' disease patients.
The standard protocol originally used involved planting the CHO-Rluc cells
from
a frozen stock, so as to seed at a concentration that produced confluent mono
layers after
18-24 hours of incubation. Initially, the Growth Medium was removed and
Stimulation
Medium was added to the monolayers, to which a series of TSH standards (e.g.,
0, 10,
100, 1000 !AU TSH/ml), and patient IgG samples were added. As this approach
yielded
poor results, an overnight Starvation or conditioning period was tested.
A Starvation period resulted in improved results with lower background values
and appeared to produce good values for the TSH standards and the test patient
samples.
An additional experimental option was also tested in which polyethylene glycol
(PEG)
was used to enhance antigen and antibody binding. In these experiments, PEG
was added
to the Stimulation Medium.
In various experiments, different media formulations and combinations were
tested, as described in the Experimental section below. For example,
starvation with the
Stimulation Medium resulted in RLU/sec values of (32,103) for the 0 OU/m1 TSH
standard, -1,148 for the 10 1.LIU TSH/ml sample, 47,478 for the 1000 'AU
TSH/ml
sample, and 19,350 for IgG sample #13. In this, and the following discussions,
the
numbers in parentheses represent the 0 p.IU TSH/ml value, which is subtracted
from the
values for the standards or samples to yield net values.
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Starvation with standard HBSS resulted in RLU/sec values of (21,671) for the 0
11/m1 TSH control, 1,336 for the 10 IU TSH/ml sample, 82,466 for the 1000 !AU
TSH/ml sample, and .39,082 for IgG sample #13. Starvation with standard HBSS
and 6%
PEG in the Stimulation Medium resulted in RLU/sec values of (32,562) for the 0
p.IU/m1
TSH control, 5,980 for the 10 [LIU TSH/ml sample, 207,831 for the 1000 WU 5
TSH/ml
sample, and 174,461 for IgG sample #13. Thus, starvation with standard HBSS
yielded
higher values for TSH and the Graves' disease samples, and the incorporation
of PEG
into the Stimulation Medium yielded even higher values. These higher values
appear to
impart a higher level of sensitivity in the methods of the present invention,
as compared
to the above described methods. Nonetheless, the long duration of these assays
involving
Starvation periods is disadvantagous. It was hypothesized that assay
improvements that
shortened the 3-4 days assay period might also improve assay sensitivity and
accuracy.
E. Chimeric TSH Receptor Cell Lines
In one embodiment, the present invention contemplate recombinant cell lines
(i.e.,
for example, CHO and RD) that express a TSH/LH/TSH chimeric receptor (i.e.,
for
example, RMc4) in combination with a firefly luciferase gene. In one
embodiment, the
expression is driven by a human glycoprotein alpha subunit promoter. Although
it is not
necessary to understand the mechanism of an invention, it is believed that by
using a
chimeric receptor, binding of the blocking antibodies (i.e., for example,
TBAb) is either
eliminated and/or reduced. In one embodiment, a chimeric receptor comprises at
least
one genetic modification such that only a TSAb binding region is expressed. It
is
believed that the recombinant cell lines have increased specificity when
compared to
either the CHO-Luc cells or 'CRONUS assay.
III. Monitoring of Immune Response Development
As indicated above, the present invention also provides methods and
compositions for the monitoring of immune response development. In particular,
the
present invention provides methods and compositions suitable for monitoring
the
response of individuals to vaccination.
In one embodiment, a pre-immune serum (i.e., serum collected prior to
administration of vaccine) may be used as a baseline for control purposes.
Such serum
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would also be collected shortly following vaccination (e.g., 1-2 weeks after
vaccination),
as well as periodically in the months following vaccination. The serum samples
are then
tested for the presence and quantity of neutralizing antibodies.
In some embodiments, diagnostic assays are conducted to monitor the response
to
viral antigens. In such assays, cells such as ELVISTM (Diagnostic Hybrids,
Athens, OH)
are used in combination with a polyethylene glycol (PEG) solution of the
present
invention. In one embodiment, PEG enhances the antigen-antibody reaction,
thereby
resulting in higher reactivity.
IV. TSI Detection In CHO-Mc4luc and RD-Mc4luc Cell Lines
In one embodiment, the present invention contemplates using genetically
engineered Chinese Hamster Ovary (CHO) and/or human Rhabdomyosarcoma cells
(RD)
for diagnosing Graves' disease and/or monitoring Graves' disease therapy.
Clinical laboratories currently utilize various cells and reaction buffer for
the
detection and measurement of stimulating autoantibodies specific to Graves'
disease in
patient sera for identifying patients suffering from this disease and
monitoring their
therapy. For example, cells comprising genetically modified CHO cells
containing wild
type human Thyroid Stimulating Hormone Receptor (TSHR) and the CRE-Luc
reporter
system are utilized by numerous laboratories. These cells, however, need one
day for
growth and one day for starvation which puts a time constraint on test results
availability.
On the third day, the patient's serum specimens are incubated with the cells
and reaction
buffer in order to detect the presence of the Graves' autoantibodies. In some
embodiments, the present invention contemplates methods that do not require
these
multi-day assay procedures. In one embodiment, these shorter methods do not
have a
Starvation period incubation. The advantages of a quick, accurate, and
sensitive assay to
diagnose Grave's disease are explained more fully below.
In one embodiment, the present invention contemplates a method for improving a
thyroid stimulating immunoglobulin (TSI) detecting cell line (CHO-RLuc). In
one
embodiment, the cell line further comprises a chimeric receptor. In one
embodiment, the
chimeric receptor comprises a human Thyroid Stimulating Hormone Receptor
(TSHR)
and a rat Luteinizing Hormone (LH) (i.e., for example, a RMc4 receptor).
Although it is
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not necessary to understand the mechanism of an invention, it is believed that
a chimeric
TSH receptor provides improved binding specificity for TSI such that a
Starvation period
in the diagnostic assay is not required.
In one embodiment, the present invention contemplates a method for expressing
the Mc4 chimeric receptor in the CHO cells and/or RD cells (or other mammalian
cells).
In one embodiment, the method further comprises using CRE-Luc as a reporter
gene to
detect TSI. In one embodiment, the chimeric receptor provides greater
specificity than a
wild-type receptor by preferentially binding to stimulating autoantibodies
(i.e., as
opposed to blocking autoantibodies). In one embodiment, the chimeric receptor
provides
greater sensitivity than a wild-type receptor by preferentially binding to
stimulating
autoantibodies (i.e., as opposed to blocking autoantibodies). In one
embodiment, the cell
culture further comprises PEG. Although it is not necessary to understand the
mechanism of an invention, it is believed that because Graves' patient sera
can have both
stimulating and blocking autoantibodies, the wild type TSH-R receptor will
bind with
both antibodies equally. Further, it is believed that blocking autoantibodies
can moderate
and suppress stimulating autoantibody activity.
These chimeric TSH-R receptors expressed in the disclosed cell lines offer the
following advantages over currently used cell lines:
1. The system results in a lower luciferase activity background leading to
higher Signal:Noise (S:N) or Signal:Background (S:B) ratios.
2. The cell lines do not need to be "starved" overnight, a requirement for
currently used cell lines in order to maximize the signal resulting from TSI
binding. This
change reduces the turn-around time from a current 3 day assay to a 2 day
assay, which is
very advantageous to the laboratory, the physician, and the patient.
3. The assay is designed to measure stimulating antibodies, whereas the
wild
type TSH-R is responsive to both stimulating and blocking antibodies whereas
this Mc4
chimeric receptor is responsive only to stimulating antibodies, thereby
providing greater
specificity for what is being measured.
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V. Chimeric TSH Receptor
In one embodiment, the present invention contemplates novel diagnostic cell
lines
that detect thyroid stimulating hormone receptor (TSH-R) autoantibody (i.e.,
for example,
thyroid stimulating immunoglobulin; TSI) with high detection sensitivity and
specificity.
In one embodiment, the cell line comprises a recombinant Chinese Hamster Ovary
cell
(i.e., for example, a CHO-K 1 cell). In one embodiment, the cell line
comprises a Human
Rhabdomyosarcoma (RD) cell.
In one embodiment, the present invention contemplates a vector comprising a
nucleic acid sequence encoding a hTSH/rLH-R fusion protein (i.e., for example,
RMc4)
linked to a firefly luciferase reporter gene and in operable combination with
a
glycoprotein hormone alpha subunit promoter. In one embodiment, a cell line is
transfected with the vector. In one embodiment, the transfected cell line
expresses a
human TSH-R/rat Luteinizing hormone (LH) chimeric receptor (hTSH/rLH-R), under
conditions such that the luciferase reporter signal is detected.
A. Chimera Construction
The identity of binding sites for TSH and thyroid stimulating autoantibodies
in
relation to Graves' disease was initially examined by constructing human/rat
chimeric
TSH-R constructs. A partial substitution of the human TSH-R with the
corresponding rat
sequence resulted in the following chimeric receptors: i) Mc1+2 substituting
amino acid
residues 8-165; ii) Mc2 substituting amino acid residues 90-165; and iii) Mc4
substituting
amino acid residues 261-370. The data suggested that amino acid residues 8-165
contain
an epitope specific for thyroid stimulating autoantibodies which are not the
same as those
required by TSH. Significant heterogeneity in the binding sites between
idiopathic
myxedema thyroid stimulating antibodies, Graves' disease thyroid stimulating
antibodies,
and TSH was observed. Tahara et al., "Immunoglobulins From Graves' Disease
Patients
Interact With Different Sites On TSH Receptor/LH/CG Receptor Chimeras Than
Either
TSH Or Immunoglobulins From Idiopathic Myxedema Patients" Biochem Biophys Res
Comm 179:70-77 (1991).
Early studies demonstrated transfection and expression of chimeric TSH
receptors
that included segments from rat TSH receptors and rat lutenizing hormone
chorionic
gonadotropin receptors. Various rat TSH amino acid sequences were substituted
with the
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corresponding rat LH/GC sequences. The data demonstrated that amino acid
residues
268-304 were not critical for generating the cAMP response but did eliminate a
TSH high
affinity binding site. Akamizu et al., "Chimeric Studies Of The Extracellular
Domain Of
The Rat Thyrotropin (TSH) Receptor: Amino Acids (268-304) In The TSH Receptor
Are
Involved In Ligand High Affinity Binding, But Not In TSH Receptor-Specific
Signal
Transduction" Endocr J40:363-372 (1993). The heterogeniety of anti-TSH
receptor
antibodies was addressed by comparing binding of: i) TSH-binding inhibitory
immunoglobulin; ii) thyroid-stimulating antibody; and iii) thyroid blocking
antibody
using a chimeric human TSH receptor wherein amino acid residues 90-165 of the
human
TSH receptor were substituted by equivalent amino acid residues from the
lutenizing
hormone chorionic gonadotropin receptor. The binding data suggest that there
might be
two different types of thyroid-stimulating antibodies, three different types
of TSH-
binding inhibitory immunoglobulins, and one nonfunctional antibody.
Chimeric TSH receptors have been reported to detect and characterize various
types of circulating antibodies suspected of having a relationship with
Graves' disease.
Such antibodies are believed to include, but are not limited to, stimulating
autoantibodies
that can activate TSH-R and blocking autoantibodies that can block TSH-R
binding by
either TSH or stimulating autoantibodies. For example, chimeras of human TSH-R
(hTSH-R) and lutenizing hormone human chorionic gonadotropin receptor (LH-hCG-
R)
included an RMc4 chimera having amino acids 261-370 of the hTSH-R substituted
with
equivalent residues from a human LH/CG-R. The ability of purified IgG samples
from
Graves' disease sera samples to stimulate cAMP production was measured by
radioimmunoassay. Kung et al., Epitope Mapping of TSH Receptor-Blocking
Antibodies
In Graves' Disease That Appear During Pregnancy" J Clin Endocrinol Metab
86:3647-
3653 (2001).
The interactions between TSH stimulating and blocking autoantibodies was
addressed by using two types of TSH-R chimera constructs. The first chimera is
designated Mc2 having human TSH-R amino acid residues 90-165 substituted by
equivalent residues from rat lutenizing hormone chorionic gonadotropin
receptor. The
second chimera is designated Mc1+2 having human TSH-R amino acid residues 8-
165
substituted by equivalent residues from rat lutenizing hormone chorionic
gonadotropin
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receptor. Evaluation of circulating autoantibodies in Graves' disease patients
showed
that blocking autoantibodies do not strongly antagonize the action of
stimualting
autoantibodies, but could be responsible for underestimating stimulating
autoantibody
activities as measured by current CHO-hTSH-R diagnostic assay methods. Kim et
al.,
"The Prevalance And Clinical Significance Of Blocking Thyrotropin Receptor
Antibodies In Untreated Hyperthyroid Graves' Disease" Thyroid 10:579-586
(2000).
The DNA sequence of the chimeric hTSH/rLH-R receptor (RMc4) contains a
total of 2,324 base pairs and encodes 730 amino acids. Figure 8. In this
chimeric
receptor, the human TSH-R region ranging from amino acid number 262 to 335 was
substituted with the corresponding 73 amino acids from the rat luteinizing
hormone (LH)
receptor
The sequence that drives the expression of the luciferase reporter is a 236
nucleotide glycoprotein alpha subunit promoter, which contains a cyclic AMP
(cAMP)
regulatory element (CRE) and was cloned by PCR. The nucleotide sequence of the
cloned promoter was determined by DNA sequencing and was confirmed by sequence
comparison with Gene bank sequence AF401991. An alignment of the cloned
promoter
with a GPH promoter amplified by PCR from HEK cells indicate that the two
sequences
are identical. Figure 9.
C. Chimera Diagnostic Assay
The response of the CHO-RMc4luc, RD-RMc4luc and CHO-Rluc cell lines to
negative and positive TSI sera was then compared. The cells were incubated
with TSI
negative and positive sera for three hours. Cells were then lysed and
luciferase activity
was measured by a Veritas Microplat Luminometer. The results indicated that
both the
CHO-RMc4luc and RD-RMc4luc cell lines had much higher detecting sensitivity
when
compared to the CHO-Rluc cells. Figures 10A, 10B, 10C, 10D and 10E. A
comparison
of the ratio of luciferase RLU from TSI positive sera to the negative sera
(ratio of SIN,)
shows that CHO-RMc4luc and RD-RMc4luc cells were 6-8 and 2.1-4 times more
sensitive than CHO-Rluc cell line. Figures 10B and 10D, respectively. CHO-
RMc4luc
cells were about 1.3 to 3.5 more sensitive than RD-RMc4luc cells. Figure 10E.
In
addition, the CHO-RMc4luc had lower levels of induced luciferase activity than
CHO-
Rluc when tested with TSI negative serum leading to lower background and
increased
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signal/negative (S/N) ratios. Figure 10A. Furthermore, both CHO-RMc4luc and RD-
RMc4luc cell lines showed very low standard deviation values. Figure 10A and
Figure
10C, respectively. A TSI positive serum, denoted #19, showed a high luciferase
induction
level on the CHO-RMc4luc, RD-RMc4luc and CHO-Rluc cell lines This serum was
diluted and tested on these cell lines to compare the sensitivities. Figure
10E.
Further, detecting sensitivity between the CHO-Rluc, CHO-RMc4luc and RD-
RMc4luc cell lines induced with a serially diluted TSI positive serum was
compared. For
example, a TSI positive serum was serially diluted and incubated on the
different cell
lines for three hours. The RD-RMc4luc and CHO-Rluc cell lines showed linear
responses of the ratio of S/N in the serum dilution range between 1:2 and 1:8.
However,
the slope of the dose response (value) and hence, the detection sensitivity,
for RD-
RMc4luc was much higher than that of CHO-Rluc cell line. Figure 11A. CHO-
RMc4luc
did not show a linear response of the ratio of S/N at no or low serum
dilutions. Figure
11B. CHO-RMc4luc cells, however, did show a linear dose response of the ratio
of S/N
from the serum dilution ranging from 1:32 to 1:128. Figure 11C. Note that the
slope of
the dose response (value) was even higher than that of RD-RMc4luc cell line.
Figure
11A versus Figure 11C.
The CHO-RMc4luc cell line was also compared to the CHO-Rluc cell line for
TSH sensitivity. The S/N ratio was derived from the luciferase assay using CHO-
Rluc,
CHO-RMc4luc and RD-RMc4luc cell lines induced with recombinant human TSH.
Recombinant human TSH at various concentrations was incubated with CHO-RMc4luc
or CHO-Rluc cell lines for three hours. After incubation, the luciferase
assays were
performed. The results indicated that they both are able to detect TSH at a
concentration
as low as 51.1.1U/ml, but the detection sensitivity of CHO-RMc4luc was much
higher than
that of the CHO-Rluc cell line. Figure 12.
CHO-RMc4luc and CHO-Rluc cell lines were also tested for their specificity
using other anterior pituitary hormones including human luteinizing hormone,
(hLH,)
human follicle stimulating hormone (hFSH) and human chorionic gonadotropin
(hCG),
all of which share a common alpha subunit. Neither cell line showed any cross
activity
with the tested hormones. Table 1.
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Table 1. Specificity of CHO-Rluc and CHO-RMc4luc to human TSH and other
hormones.
A. Luciferase S/N Ratio Comparision Of TSI Serum To Gonadotropin Hormones
Ratio of Signal/ Negative
FSH LH HCG hTSH
Positive TSI serum (364mIU/m1) (455 mIll/m1) (29.51U/m1)
(76 1U/m1)
CHO-Mc4luc 9.4 0.6 0.9 0.8 19.4
CHO-Rluc 1.9 0.7 0.8 0.96 2.9
B. CHO-RMc4luc Cell Line Results
CHO-RMc4luc -TSI serum + TSI serum Hormones
RLU 1589 15069 1019
FSH (364 mIU/m1) Ratio of S/N 9.5 0.6
_
RLU 1737 15565 1561
LH (455 mIU/m1) Ratio of S/N 9 0.9
_
RLU 1432 13491 1168
hCG (29.51U/m1) Ratio of S/N 9.4 0.8
RLU 1284 12512 24880
hTSH (76 IU/m1) Ratio of S/N 9.7 19.4
C. CHO-Rluc Cell Line Results.
CHO-Rluc -TSI serum + TSI serum Hormones
RLU 1052 1929 728
FSH (364 mIU/m1) Ration of S/N 1.8 0.7
RLU 1058 2011 835
LH (455 mIU/m1) Ration of S/N 1.9 0.8
RLU 946 1976 912
hCG (29.51U/m1) Ration of S/N 2.1 0.96
RLU 847 2291 2495
hTSH (760U/m1) Ration of S/N 2.7 2.9
CHO-RMc4luc and CHO-Rluc cell lines were used to screen normal human sera
to determine the distribution of the ratio of S/N derived from a luciferase
assay.
Comparisons of distribution of the S/N ratios derived from luciferase assays
on CHO-
37
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Rluc and CHO-RMc4luc cell lines induced with sera from 108 normal people were
performed. All serum samples were tested in both CHO-RMc4luc and CHO-Rluc cell
lines. A known normal serum was used as a reference for calculating S/N
ratios. The
distribution of CHO-RMc4luc cell line revealed a pattern very similar to that
of the CHO-
Rluc cell line. The mean of the CHO-Rluc cell was 1 and the CHO-RMc4luc was
0.88.
The standard deviation of CHO-Rluc was 0.23 and CHO-Luc was 0.21. Figure 13.
The responses of the CHO-RMc4luc, RD-RMc4luc and CHO-Rluc cell lines to
clinical patient serum samples were compared. The ratio of S/N derived from
the
luciferase assay on CHO-Rluc, CHO-RMc4luc and RD-RMc4 luc cell lines induced
with
12 clinical serum samples. Each of the 12 serum samples was tested in the CHO-
RMc4luc, RD-RMc4luc and CHO-Rluc cell lines. Luciferase activities of these
samples
were compared to that from a known negative serum sample (negative reference).
The
results of this study indicated that both the CHO-RMc4luc and RD-RMc4luc cell
lines
had much higher detection sensitivity when compared to the CHO-Rluc cell line
with the
and CHO-RMc4luc cell line being the most sensitive cell line. Figure 14.
VI. Chimeric Receptor Assay And The Starvation Pre-Conditioning Period
As disscussed above, the CHO-RMc4luc cell line provides definite advantages in
sensitivity and specificity over the currently used CHO-Rluc cell lines. For
example, the
CHO-RMc4luc cells, upon stimulation by Graves' Disease antibodies, provide
increased
luciferase responses as measured by the Relative Light Units (RLU) output.
This
improvement allows the elimination of the one day starvation step from the
protocol.
The standard procedure used for the CHO-Rluc cell line includes a starvation
period. The protocol for the Starvation format is to plant the cells for
Growth on day 1,
Starve on day 2 and Stimulate and measure RLU on day 3. When the starvation
period is
eliminated, the "starve day 2" step is eliminated and the final results can be
reported to
the physician on day 2 instead of day 3, which is much more desirable. This
non-starved
protocol provides a significant advantage to the user and physician because
labor for the
user is significantly reduced and the physician can have the results the next
day, all with
an assay of higher accuracy than that provided by the CHO-Rluc protocol with
its one
day of starvation.
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Starvation periods were used to increase the RLU or % separation between
Normal and Graves' Positive sera. This RLU separation relates directly to the
accuracy
of the assay. The effect of the Starvation period was tested using Graves'
Positive and
Normal serum specimens using the two different cell lines, CHO-Rluc and CHO-
RMc4luc. See, Table 8.
Table 8: Relative Effects Of Starvation Periods On CHO-Rluc And CHO-RMc4luc
Cells
' - =
Cutoff = . ^ , , -Cutoff .. - '1 .. = ' '
Cutoff
, . . , ....._ ,.,. ..., ..
...... . .. . , , ..
130% 140% - 170%
614,0436.16,-ir:. '*:: diii.O.F4A.464; .,.; :. CHQ-RMO4
Serum # h _ ,_ ;[, jarveScIA: :!, ..":;:'.;NOTI72 tarke,d-
,:!: . , '.'::'. . . Starved -, I
(SRR)% RLU (SRR)% RLU (SRR)% RLU
26 118% 874 310.2% 13457 1023.3% 10162
27 149% 1105 208.3% 8751 198.0% 1966
28 302% 2236 525.6% 22083 1291.5% 12825
29 504% 3725 627.3% 27211 1912.2%
18988 0
30 380% 2811 481.4% 20885 1366.7%
7763 >
31 300% 2221 660.3% 27743
2345.7% 13323 ._
32 571% 4226 525.9% 22096
1855.6% 10540 .._,
33 . 144% 1062 405.8% 17049 1495.9% 8497
--
34 208% 1537 150.5% 6530 265.8% 1510
35 167% 1234 353.7% 15345 917.3%
5210 (I)
0
36 180% 1334 574.2% 24909
2340.0% 13291 0-
37 119% 878 187.5% 7878 388.9% 2209
38 114% 846 231.9% 9745 468.6% 2662
39 104% 771 145.8% 6127 287.8% 3031
40 137% 856 81.0% 3405 96.0% 1011
Ave. 1714 15548 7532
7 94% 504 39.2% 1944 80% 525
8 90% 486 48.1% 2382 88% 658
9 89% 482 38.1% 1887 78% 724
110% 591 37.8% 1872 79% 639
12 105% 566 51.1% 2290 90% 963
14 113% 611 43.1% 1930 99%
907 -
16 110% 591 35.9% 1608 75%
659 CU
E
17 82% 443 36.8% 1648 85% 617
18 108% 582 40.5% 1816 77% 696
19 98% 542 42.6% 1908 84%
666 0
21 78% 431 84.4% 3781
101% 613 Z
23 90% 495 55.9% 2504 49% 587
24 125% 693 35.9% 1609 82% 428
25 98% 541 50.8% 2274 70% 711
26 107% 591 45.7% 2049 75% 603
27 104% 577 62.8% 2813 71% 647
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28 109% 600 38.1% 1707 59% 613
29 96% 532 48.2% 2021 83% 509
30 87% 481 39.5% 1655 87% 721
31 105% 489 40.4% 1696 66% 769
32 75% 350 51.8% 2173 67% 589
33 89% 416 78.4% 3289 62% 592
Ave. 527 2130 656
+RLU/-RLU 3.25X 7.3X 11.9X
The RLU values for Graves' Positive and Negative sera as obtained using the
respective protocols for starved CHO-Rluc and CHO-RMc4luc cells, and non-
starved
CHO-RMc4luc cells. The intended effect of starvation on CHO-RMc4luc cells was
to
decrease the background level of luciferase activity in the cells, thereby
raising the ratio
of RLU between Graves' Positive and Graves' Negative sera. For these sets of
sera, the
average ratio for Starved CHO-Rluc is 3.25x, for non-Starved CHO-RMc4luc is
7.29X
and for Starved CHO-RMc4luc cells is 11.5X.
Thus, the non-starved CHO-RMc4luc cells provide a greater than 2-fold increase
in luminosity (i.e., and therefore sensitivity) over the starved CHO-Rluc
cells. This
provides distinct advantages of a protocol lasting one day shorter and
providing more
accurate and sensitive results. The Serum:Reference Ratios (SRRs) compare the
RLU
values as percentages and further confirm that the CHO-RMc4luc cells (whether
starved
or non-starved) provide an improved separation between the RLUs between Normal
and
Graves' Disease sera. The Cutoff values presented are approximate and are
indicative of
Graves' Disease when the assayed value is > to the cutoff value of the
particular
protocol/cell line.
VII. Chimeric Receptor Assay Improvements With Glucocorticoids
Because the above data indicate that Starvation periods also provide an
improvement of the accuracy and sensitivity of the CHO-RMc4luc cells, further
investigations were then directed to develop superior CHO-RMc4luc cell assays
without
a Starvation period.
One set of data was collected in accordance with Example 15, wherein CHO-
RMc4luc cells underwent Starvation periods and then were incubated with
various
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concentrations of dexamethasone (DEX). The data clearly indicate that
dexamethasone
significantly improved the RLU intensity over and above that provided by a
Starvation
period alone. Table 9.
Table 9: Effect of Dexamethasone On Starved CHO-R-Mc4 Cells
CHO-RMc4luc
DEX Reference Serum
uM 0 12. 25 40 50 100 pc*
Test 1 151 141 132 126
122 973 1601
Test 2 148 118 121 128 117 ,
118 1277
Test 3 122 129
129 123 135 120 1306
Avg 140 129 127 126 125 112 1395
S/B 9.9
DEX Patient #18 Serum
uM 0 12.5 25
40 50 100
Test 1 2795 4710
5172 5039 4861 3542
Test 2 3393 5456
5151 5198 5298 3414
Test 3 3167 5330
5260 5038 4909 3521
Average 3118 5165 5194 5092 5023 3492
S/B 22.2 39.8
40.6 40.4 40.1 31.1
ok 100 180
183 182 181 140
of S/B
* - Positive Control
The data show that dexamethasone reduces the Reference serum RLU readings
while at the same time greatly increasing Patient #18 RLU readings. Overall,
the
presence of dexamethasone results in about an 80% increase in S/B ratios for
the Patient
#18 serum samples.
These observations provided a suggestion that an improved assay may result if
dexamethasone is used in place of a Starvation period. Consequently, a
comparison
between cells exposed to a Starvation period and cells only exposed to
dexamethasone
(40 pM) in the Growth Medium was performed. See Example 16. To compare the
different protocols, the RLU results for Graves' Positive and Graves' Negative
serum
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were averaged together and their respective percentages above the Reference
standards
were calculated. Table 10.
Table 10: Comparison Of Starved vs. Dexamethasone Treated CHO-rMc4 Cells
11111 .
¨ CHO-Mc4 Non-Starvation Protocol 4 CHO-Luc
Protocol (Starvation)
.=,deMc4wIOex Mc4 w/o Dee' \ Luc M54,W/ Oex Mc4 wlo Dex Luc
12555 1103 177 4978 4866 24?t3
=
1951 1895 100%, 1 445 492 749
qn--µ,õD "2'3436 15827 972 c 957 1129 691
ykõ CHO-Mc4 Non Starvation PnitOi0c4 CHO-Luc
Protocol (Starvation)
t xt,k , Mc4 w/ Dex Mc4 w/o, &X ,! _LUC; <;, Mc4
w/ Dex Mc4 w/o Dex
iLuc .%.
alio? )7,; ratar. 189./p, 17/9;%/ 834% 431%
'4FTN-eijat.777(n-77' 58% Ai32%pA107% 46% 44% 108%
100% -
100% 1al
100% 100% 100% 100%
The data demonstrate that non-starved CHO-RMc4luc cells with dexamethasone
provides a more sensitive (and therefore more accurate) detection of
circulating TSI's as
compared to non-starved CHO-RMc4luc cells without dexamethasone. For example,
in
the Graves' Positive patient serums not subjected to a Starvation period, CHO-
rMc4luc
cells with dexamethasone showed a 365% increase in luminescence (as relative
to the
Reference) while CHO-rMc4 cells without dexamethasone showed a 189% increase
in
luminescence.
The improvement in sensitivity and accuracy with dexamethasone is even more
dramatic when comparing the Grave's Positive patient serums with the Grave's
Negative
patient serums. For example, in the presence of dexamethasone the difference
between
Grave's positive and Grave's negative serums is 309% (i.e., for example,365% -
56%)
but in the absence of dexamethasone the difference between Grave's positive
and Grave's
negative serums is 157% (i.e., for example, 189%-32%).
These results clearly show that dexamethasone provides a better assay system
in
terms of sensitivity due to a stronger luminescent signal strength. Such an
improvement
results in improved testing accuracy and when combined with the RMc4luc
testing
platform, the present invention contemplates a diagnostic assay that is more
rapid and
accurate than any previously disclosed TSI antibody assay requiring a
Starvation medium
peroid.
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Further studies demonstrated that this effect was not limited to dexamethasone
but
can be expected from most, if not all, glucocorticoids. For example, the data
presented
herein show that other glucocorticoids also improve the sensitivity of the CHO-
RMc4luc
assay to provide equivalent sensitivity in comparison with substitution for a
Starvation
medium period. Nonetheless, the presence of a glucocorticoid provides the
advantage
that the assay can be performed in two days, rather than three days.
Alternative glucocorticoids (GCs) where compared to dexamethasone (40 04) on
the basis of Relative Light Units (RLUs) and Serum Reference Ratios expressed
in
percentages (SRRs%). For this purpose, all five (5) tested glucocorticoids
show a generic
effect in improving the sensitivity of the RMc4luc assay in the absence of a
Starvation
period.
The data was collected in accordance with the protocol outlined in Example 17.
The data was calculated as the difference (A) in RLU values or SRR% values for
each
glucocorticoid (GC) concentration by subtracting the RLU value or SRR% value
for the
Normal control at that concentration from the RLU value or SRR% value for the
Positive
control at that concentration, respectively.
The data demonstrate that all four GCs improve signal intensity by at least
6000
RLUs, wherein hydrocortisone and cortisone have signal intensities equivalent
to
dexamethasone. See, Figure 17. When the data was calculated as SRR%, however,
it
can be seen that dexamethasone improves signal-to-noise ratio by approximately
2-fold
when compared to all the alternative GCs. See, Figure 18. Nonetheless, the
data
demonstrate that any glucocorticoid can provide improvements in RMc4luc assay
sensitivity and accuracy such that a Starvation medium period is not required.
VII. Kits
In yet other embodiments, the present invention provides kits for performing
Graves' disease diagnostic assays using chimeric TSH receptors. The kits
preferably
include one or more containers containing a cell line-based diagnostic method
of this
invention. In some embodiments, the containers may contain a glucocorticoid
including,
but not limited to, dexamethasone, cortisone, hydrocortisone, prednisone, or
fluticasone.
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In some embodiments, the kits contain all of the components necessary or
sufficient for
performing a Grave's disease diagnostic assay to detect circulating TSH
autoantibodies in
patient sera, including all controls, directions for performing assays, and
any software for
analysis and presentation of results. In some embodiments, the kits contain
vectors
encoding chimeric TSH receptors capable of transfecting cell lines. In some
embodiments, the kits comprise all materials necessary or sufficient to
perform diagnostic
assays in a single reaction and provide diagnostic, prognostic, or predictive
information
(e.g., to a researcher or a clinician). For example, such a kit might contain
a cell line
comprising a chimeric TSH receptor and a luciferase reporter system. In some
embodiments, the kits comprise one or more of a vector comprising a first
nucleic acid
sequence for an Mc4 chimeric TSH receptor, a second nucleic acid sequence for
a
luciferin/luciferase reporter system, and a third nucleic acid sequence for a
promoter.
Other embodiments also include buffers, control reagents, detection devices,
software,
instructions, and TSH autoantibody standard preparations.
The kits may also optionally include appropriate systems (e.g. opaque
containers)
or stabilizers (e.g. antioxidants) to prevent degradation of the reagents by
light or other
adverse conditions. Each solution or composition may be contained in a vial or
bottle
and all vials held in close confinement in a box for commercial sale.
The kits may optionally include instructional materials containing directions
(i.e.,
protocols) providing for the use of the reagents in the diagnosis, detection,
and/or
treatment of Graves' disease. While the instructional materials typically
comprise written
or printed materials they are not limited to such. Any medium capable of
storing such
instructions and communicating them to an end user is contemplated by this
invention.
Such media include, but are not limited to electronic storage media (e.g.,
magnetic discs,
tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such
media may
include addresses to interne sites that provide such instructional materials.
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Experimental
The following examples are provided in order to demonstrate and further
illustrate
certain preferred embodiments and aspects of the present invention and are not
to be
construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations
apply:
eq (equivalents); M (Molar); ;AM (micromolar); N (Normal); mol (moles); mmol
(millimoles); [tmol (micromoles); nmol (nanomoles); g (grams); mg
(milligrams); jig
(micrograms); ng (nanograms); or L (liters); ml (milliliters); p.1
(microliters); [LIU (micro
International Units); cm (centimeters); mm (millimeters); pm (micrometers); nm
(nanometers); C (degrees Centigrade); sec. or s (second(s)); min. and m
(minute(s));
MW (molecular weight); thyroid stimulating hormone or thyrotropin (TSH); bTSH
(bovine TSH); TSI (thyroid stimulating immunoglobulin); TSAb (thyroid
stimulating
antibodies); EDTA (ethylene diamine tetraacetic acid); RLU/sec (relative light
units per
second); GM or PM (Growth Medium or Planting Medium); SM (Starvation Medium);
HBSS (Hank's Balanced Salt Solution); EMEM (Eagle's Minimum Essential Medium);
FBS or FCS (fetal bovine serum or fetal calf serum); DMSO (dimethyl
sulfoxide); CHO
(Chinese hamster ovary cells); CHO-R (CHO cells transfected with the human TSH
receptor; CHO-Rluc (CHO-R cells transfected with the cre-luciferase reporter
gene
complex); Oxoid (Oxoid, Basingstoke, England); BBL (Becton Dickinson
Microbiology
Systems, Cockeysville, ME)); DIFC0 (Difco Laboratories, Detroit, Nil); U.S.
Biochemical (U.S. Biochemical Corp., Cleveland, OH); Fisher (Fisher
Scientific,
Pittsburgh, PA); Sigma (Sigma Chemical Co., St. Louis, MO.); ATCC (American
Type
Culture Collection, Rockville, Maryland); LTI (Life Technologies, Rockville,
MD); and
Promega (Promega Corp., Madison, WI).
In the following methods, all solutions used in these methods were sterile
(with
the exception of TSH, controls, patient specimens) and treated aseptically.
All
manipulations were conducted in a biosafety cabinet under aseptic conditions.
Cell
culture media (e.g., Ham's F-12, EMEM, etc.) were obtained from LTI, while
additive
reagents such as non-essential amino acids were obtained from Sigma.
Freezer vials of cells should not be allowed to warm from their -80 C (or
lower)
storage temperature until immediately prior to thawing and use in the methods
of the
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present invention, as cycling of the temperature may result in viability
losses. Because it
contains dithiothreitol, which is unstable at room temperatures, the 5x cell
lysis solution
should be removed from its -20 C storage temperature only long enough to
remove the
required volume for preparation of the lx solution. As it also contains
dithiothreitol,
reconstituted luciferase substrate solution should be kept frozen at -20 C
until just prior
to use, at which time it may be removed and placed in a 25-37 C water bath to
thaw and
reach room temperature.
In general, when removing liquid from wells (e.g., microtiter plates, etc.),
the
liquid may be dumped from the wells into a receptacle in a biosafety hood. The
residual
liquid can be drained and removed by placing the plate upside down on a
sterile,
absorbent wipe. Or, the liquid may be removed by aspiration using a fine tip
on the
aspirator. If aspiration is used, the plate is held at a steep angle so that
the liquid does not
overflow the wells, and the aspirator tip is directed down the side of the
well almost to
the bottom to remove the liquid and only leave minimal residue. However, care
must be
exercised in order to prevent disturbance of the cell monolayer, as the cells
can be easily
removed by the aspirator.
As indicated in the methods below, it is recommended that specimens,
standards,
and controls be run in triplicate. Because of the viscous nature of Solution 3
and the
difficulty in achieving adequate mixing in the wells, the best reproducibility
was
achieved when the total triplicate volume is +10% (33 1) of these reagents is
transferred
to the required triplicate volume +10% (330 I) of Solution 3, thoroughly
mixed, and 110
I transferred to the triplicate wells.
In the preparation of cell monolayers (e.g., within the wells of microtiter
plates), it
is preferred that the cells be distributed evenly within the wells. Thus, in
order to avoid
uneven cell distributions, the transfer of cell suspensions into wells should
be performed
in a vibration-free biosafety hood. After all of the wells in a plate have
received cells, the
plate is covered and carefully placed on a solid, vibration-free surface, for
30 minutes, to
allow the cells to attach undisturbed, to the bottom of the wells. This helps
ensure that an
even distribution of cells is present in each. of the wells.
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EXAMPLE 1
Preparation of CHO-Rluc Cells For Testing
In these experiments, CHO-Rluc cells were prepared from W-25 CHO-R cells for
use in the testing methods to detect TSI in Graves' disease patients. Pools of
puromycin-
resistant cells were obtained and tested for light output in response to
bovine TSH.
Clones with the highest light output were selected for use in the experiments
described
below.
CHO-Rluc cells were grown in cell culture flasks (e.g., T-225 flasks) in
growth
medium containing Ham's F-12 medium, 10% FBS (heated at 56 C for 30 minutes to
inactivate complement), 2 mM glutamine, and lx non-essential amino acids. The
flasks
were incubated at 35-37 C, in a humidified atmosphere, containing 5% carbon
dioxide.
After the cell cultures reached confluence, the medium from each flask was
aspirated, and the cell monolayers were washed with HBSS without Ca and Mg.
Then,
7 ml of a 0.25% trypsin/1 mM EDTA solution were added to each flask, and
allowed to
react with the mono layers for approximately 5-10 minutes at room temperature,
in order
to detach and disperse the cells in a nearly unicellular suspension. The cell
suspensions
were then centrifuged for approximately 5 minutes at 300-400 x g. The
supernatants were
then removed and the pelleted cells resuspended in 8 ml of a medium prepared
by mixing
4 ml EMEM containing lx HBSS and 20% FBS with 4 ml of cryoprotective medium
(EMEM containing lx HBSS and 15% DMSO).
An aliquot of each cell suspension was then used to determine the number of
cells
present in the suspension. This determination can be accomplished using any
method
known in the art, including but not limited to methods using a hemocytometer
to
determine the cell count. Thus, it is contemplated that any method can be used
to
determine the cell count in the suspensions. Based on the number of cells in
the
suspension, the cells were aliquoted by volume to approximately 2 x 106 cells
into
standard freezer vials. The cells were then stored frozen at -90 C for short-
term storage.
For long-term storage, the cells were stored in liquid nitrogen (about -200
C).
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EXAMPLE 2
CHO-Rluc Assay Plate Preparation and Testing
In these experiments, CHO-Rluc cells prepared as described in Example 1 were
used in assays for diagnosis of Graves' disease. To prepare 24 monolayers for
testing, 24
wells in a 96-well microtiter plate were first treated by adding 50-100 1
0.1% gelatin
solution (Sigma) to enhance attachment of the cells to the bottom of the 24
wells chosen
for the test. Following incubation for approximately 1 minute at room
temperature, the
gelatin solution was removed from each of the wells by aspiration. It was
noted that the
gelatin can remain on the wells for longer than one minute. The gelatin serves
to coat the
wells with collagen, so that the cells attach more quickly to the wells and
reach
confluence more rapidly. However, cells can be planted and grown to confluence
without
gelatin and still perform well.
A freezer vial of CHO-Rluc cells produced as described in Example 1 was
rapidly
thawed in a 37 C water bath to provide approximately 0.4 ml cell suspension,
which was
well-mixed using a pipette. The cells were then added to 2.5 ml GM (also
referred to as
"Planting Medium"), thoroughly mixed by vortexing for 1-2 seconds, and 100 I
aliquots
of the cell suspension were added to each well, and the plates were covered.
It is
preferable to produce an even distribution of cells in each well. Thus, to
avoid uneven
cell distributions, the microtiter plate should be placed in a vibration-free
hood for cell
planting and attachment of cells to the walls of the microtiter plate. The
planted cells
were then incubated at 35-37 C, in a humidified atmosphere, containing 5% CO2,
for
approximately 20-24 hours, to allow the cells to form a nearly or completely
confluent
monolayer.
The GM was then aspirated from each well as completely as possible, being
careful not to disturb the monolayers (i.e., confluent monolayers remain in
the wells).
The monolayers were rinsed with approximately 100 111 Starvation Medium (HBSS
containing Ca ++ (0.14 g/L) and Mg ++ (0.048 g/L) per well. The Starvation
Medium was
aspirated and a fresh 100 I of Starvation Medium was then added to each well.
It is
important that these steps be conducted sufficiently rapidly that the cell
monolayers do
not dry. The plates were then incubated overnight in a 35-37 C, 5% CO2,
humidified
incubator. Following incubation, the Starvation Medium was aspirated from the
wells,
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using care to avoid disturbing the monolayers. Then, approximately 100111
Stimulation
Medium were added to each monolayer, again working quickly so that the
monolayers
did not dry.
Then, in an alternative method to that previously described, 10 p1 of patient,
control, and TSH standard solutions were added to the appropriate wells. The
TSH
standards and IgG samples were diluted with diluent (i.e., HBSS--NaC1 + 222 mM
sucrose). The TSH standards were tested at concentrations of 0, 10, 100, 1000,
and 5000
Patient samples were diluted to a concentration of 10 mg protein/ml for use in
the
assay. As the Stimulation Medium is viscous, thorough mixing of the
suspensions was
important. Adequacy of the mixing was ascertained by microscopic examination
of the
monolayers. The plates were incubated for 4 hours at 35-37 C in a 5% CO2,
humidified
incubator. The medium was carefully aspirated from each well and 150 pl lysis
solution
(Promega) was added to each well. The lysis solution contained 25 mM Tris-
phosphate,
pH 7.8, 2 mM diaminocyclohexane tetraacetic acid (CDTA), 2 mM dithiothreitol
(DTT),
10% glycerol, and 1% Triton X-100. The plates were then incubated for 30
minutes at
room temperature, to allow the monolayers to lyse. Following lysis, each well
was
scraped and stirred using a pipet tip. Then, 25 pl of lysate were removed from
each well
and placed in a luminometer tube (12x75 mm, polypropylene), and 50 p.I of
luciferase
substrate (Promega) were then added. The tubes were vortexed for 1-2 seconds
and the
RLU/sec values determined, using settings of 5 seconds delay and 10 second
read. To
obtain average net values, the average of the "0 TSH" (i.e., the negative
control) samples
was subtracted from all test average values.
EXAMPLE 3
Preparation of IgG Samples
In these experiments, patients' IgG was prepared for testing in the present
methods. Lyophilized IgG samples from 38 well-known and characterized,
untreated
Graves' disease patients were kindly provided by Dr. B.Y. Cho (Department of
Internal
Medicine, Seoul National University, College of Medicine, Seoul, Korea). As
most of the
samples had been previously tested in standard methods using CHO-R and FRTL-5
cells,
these test results were known for 35 of these samples.
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In preparation for lyophilization, the IgGs were affinity-purified using
protein A-
Sepharose CL-4B columns, as known in the art, and then dialyzed against 100
volumes of
distilled water at 4 C. The dialysis water was changed every 8 hours over a 2
day period.
After removal of denatured protein by centrifugation at 1500 xg for 15 minutes
at 4 C,
the IgG was lyophilized and stored at -20 C until used in the experiments
described
herein.
In some experiments, purified untreated Graves' IgG was diluted in normal
serum
(euthyroid sera discussed in Example 7, below), and assayed using the CHORluc
assay
described below.
EXAMPLE 4
CHO-Rluc Assays
In these experiments, the performance of CHO-Rluc cells using the method
described by Evans et al. (Evans et al.,J. Clin. Endocrinol. Metabol., 84:374
(1999)) was
evaluated. The media from the cell mono!ayers in the 24 wells used in the 96-
well
microtiter plates prepared as described in Example 2 were aspirated and
replaced with
100 I Ham's F-12 medium containing 10% charcoal-stripped calf serum (Sigma),
and
incubated overnight at 35-37 C, in a humidified atmosphere containing 5% CO2.
Then, 10 IA of bovine TSH standards diluted to a range of concentrations
(e.g., 0
10, 100, and 1000 !AU) and Graves' IgG (dissolved to a concentration of 10 mg
protein/ml in charcoal-stripped calf serum) were added to respective
quadruplicate wells.
The suspension in each well was mixed, and the plates were incubated for 4
hours at 35-
37 C, in a humidified atmosphere containing 5% CO2. The medium was then
aspirated
from each of the wells, and 150 I of lysis buffer (Promega, as described
above) were
added to each well. The plates were then incubated at room temperature for 30
minutes to
allow lysis of the cells in the wells. Then, 25 1 of each lysate were
transferred to a 12x75
polyethylene luminometer tube, to which 50 I of luciferase substrate
(Promega) were
added immediately prior to mixing and reading in the luminometer at settings
of 5
seconds delay and 10 second read. The luminometer read out provided results as
relative
light units per second (RLU/sec). The negative or "zero" TSH standard value
was
subtracted from each of the readings. In one run, the average net value for
the zero U/m1
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TSI standard was 68,011 RLU/sec, while the result for the sample containing 10
IU/ 1
was 4031 RLU/sec, the sample containing 1000 ptIU was 222,801 RLU/sec, one
Graves'
IgG test sample was 384 RLUTsec (sample #1), and another Graves' IgG test
sample was
-3012 RLU/sec (sample #9).
The Graves' IgG sample #1 and sample #9 were previously assayed using
standard FRTL-5 cells and a cAMP RIA assays. In the cAMP assay, values greater
than
153 with FRTL-5 cells are considered positive for the presence of TSI. The
cAMP value
with FRTL-5 cells for sample #1 was 212, and the cAMP value for sample #9 was
803.
The CHO-R values for these same samples (#1 and #9) were 116 and 1733,
respectively,
in an assay system where CHO-R values greater than 173 are considered to be
positive
for Graves' disease. Thus, these results clearly indicate that there is a
discrepancy
between the results obtained using different cell lines for the detection of
Graves' disease.
Indeed, the use of the Evans et al. method yielded negative results for both
IgG samples,
indicating that this system with CHO-Rluc is useless for detecting human TSI,
despite the
fact that the response to bovine TSH was very good.
Furthermore, during the development of the present invention (as described
below), it was determined that if the CHO-Rluc cells were planted in a medium
containing charcoal-stripped calf serum for 24 hours (i.e.,to reach
confluence), the cells
simply attached to the bottom of the wells, but did not multiply and become
confluent
during the incubation period, unlike the situation in which normal FBS was
used. Thus,
this surprising result indicates that the use of charcoal-stripped serum in
the medium
resulted in a starvation step for the cells, somewhat analogous to the
incubation of FRTL-
5 cells in 5H medium.
In some experiments purified, untreated Graves' IgG diluted in normal serum,
were tested in the CHO-Rluc assay (with PEG). For IgG #10, (2 mg/ml), the
RLU/sec
value was 131,461; for IgG #15 (2 mg/ml), the RLU/sec value was 180,327; for
IgG #27
(5 mg/ml), the RLU/sec value was 179,777; and for IgG#32 (5 mg/ml), the
RLU/sec
value was 112,627. These results clearly show that the CHO-Rluc assay measures
TSI in
the presence of serum.
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EXAMPLE 5
Development of Media Formulations
In view of the previously-described experiments, the effects of different
media
fornlulations were investigated for use with the CHO-Rluc cells in the
measurement of
bovine TSH and human TSI. In these experiments, various media formulations
were
tested for the "starvation," and "stimulation" steps in the CHO-Rluc assay,
using bTSH
standards and IgG extracted from the sera of Graves' disease patients.
In these experiments, once the cell monolayers contained within the wells of
96-
well microtiter plates (as described above), reached confluence, the Growth
Medium was
removed by aspiration and 100 I of Starvation Medium were added to each
monolayer.
The plates were then incubated for 16-24 hours at 35-37 C, in a humidified
atmosphere
containing 5% CO2, to starve or condition the cells. The Starvation Medium was
then
aspirated from the wells.
To perform the assay, 10 I of the patient specimen IgG, bTSH standards, and
IgG controls (normal and Graves' disease sera), were added to the monolayers
in
triplicate. The suspensions were mixed within each well, and incubated under
the above
conditions for 4 hours. The liquid was then removed from each monolayer by
aspiration,
and 150 1 of lysis buffer (Promega, as described above) were added to each
well. The
plates were allowed to incubate at room temperature for 30 minutes to lyse the
cells in the
monolayers.
In order to measure the amount of cell stimulation caused by the TSH standard
or
antibody to the TSH receptor, the luciferase in the cell lysates was measured
by adding
I of lysate to a luminometer tube to which 50 1 of substrate solution
(Promega) were
added. The suspensions were mixed and then read in a luminometer with settings
of a 5
25 second delay and a 10 second read, to determine the RLU for each sample.
In order to use the cells for TSI or TSH stimulation, the Starvation Medium
was
removed by aspiration, and 100 I of the Stimulation Medium were added to each
well.
This Stimulation Medium was HBSS--NaCl + 222 mM sucrose. The following Table 2
rovides a comparison of the formulations of HBSS--NaC1 + 222 mM sucrose and
standard HBSS.
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Table 2. HBSS Medium Formulation Comparisons
Component HBSS - - NaC1+ 222 mM
Standard HBSS (g/L)
Sucrose (g/L)
CaC12 0.144 g/L 0.14 g/L
KC1 0.373 0.400
KH2PO4 0.060 0.060
MgSO4 0.048 0.048
Na2HPO4 0.097 0.048
NaHCO3 0.00 0.35
NaC1 0.00 8.00
D-Glucose 1.00 1.00
Sucrose 76.00 0.00
HEPES 4.77 0.00
Bovine Serum Albumin 10.00 0.00
This Stimulation Medium formulation is a formulation that is commonly used in
the measurement of TSI in FRTL-5 and CHO-R cells.
The results of experiments to test various Starvation Medium formulations are
indicated in the following Table 3. In these experiments, the HBSS--NaC1 + 222
mM
sucrose Stimulation Medium was used. As indicated in Table 3, the standard
HBSS with
20 mM sucrose yielded the best signal to noise ratio (i.e., the lowest
background and
highest value for Graves' IgG).
=
Table 3. RLU/Sec Results for Various Media: Growth Versus Starvation
Medium RLU/Sec
0 TSH 10 IU 1000 ptIU #13
TSH/ml TSH/ml IgG
CHO GMa (66,232) 782 265,195
5,144
CHO Charb (50,638) 5,602 229,492
34,042
HBSS--NaC1 + 222 mM (32,289) 2,188 142,666
30,640
Sucrosec
Standard HBSS with 20 mM (27,139) 14,390 156,548
53,994
Sucrosec
a CHO GM is CHO Growth Medium containing 10% FBS.
b CHO Char. is CHO Growth Medium with 10% charcoal-stripped calf serum.
A Starvation Medium
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EXAMPLE 6
Use of PEG
As PEG may be used in in vitro antigen/antibody reactions to assist or enhance
the reaction rate, a trial was conducted in which PEG was incorporated into
the
Stimulation Medium. As this compound may decrease the off-rate or dissociation
of the
antigen/antibody complex, the use of PEG in the methods of the present
invention was
investigated.
Preliminary results with 12% PEG-8000 (i.e., ave. MW 8,000) in HBSS--NaC1
sucrose, resulted in monolayers with increased spaces between the cells. To
reduce this
apparent osmotic stress, 6% PEG-8000 in HBSS--NaC1 + 111 mM sucrose was
tested. In
these experiments, the Starvation Medium yielding the best results (i.e.,
standard HBSS +
mM sucrose) was used. The results are shown in Table 4, below.
Table 4. RLU/Sec Results for Stimulation Media With and Without PEG
Stimulation Medium RLU/Sec
0 TSH 1001 1000p,IU
#13 IgG
TSH/ml TSH/ml
HBSS--NaC1 + 222 mM Sucrose (21,671) 1,336 82,466
39,082
HBSS--NaC1 + 111 mM Sucrose + (32,562) 5,980 207,831
174,461
6% PEG-8000
As indicated in Table 4, the incorporation of 6% PEG-8000 significantly and
substantially enhanced the luminescent signal from the CHO-Rluc cells, in
response to
added bTSH, as well as Graves' IgG.
An additional experiment was conducted to determine the optimal concentration
of PEG-8000 to use in the Stimulation Medium. The net values for one Graves'
sample
(Graves' IgG #20), with an FRTL-5 cAMP value of 957, are shown in Table 5. As
indicated in Table 5, 6% PEG yielded maximum signal for Graves' TSAb.
Table 5. RLU/Sec Results for Various PEG Concentrations
Results % PEG In Stimulation Medium
2% 4% 6% 8% 10%
RLU/sec 15,566 52,259 87,908 73,260 47,991
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Subsequent experiments have shown that the Starvation Medium need not contain
20 inM sucrose, as there is no statistically significant difference in the
results with or
without it.
In addition, experiments were conducted to demonstrate that the assay of the
present invention measures thyroid-stimulating immunoglobulin in a dose-
dependent
manner. In these experiments, three Graves' disease IgG samples (#6, #11, and
#16) were
tested. Serial 3-fold dilutions were made using the Stimulation Medium
containing 6%
PEG-8000, and the methods described above. The results are shown in Figure 1,
which
shows the linearity of the dilutions. The IgG samples were prepared from 10
mg/ml
stocks, which were then tested undiluted, and serially diluted (3-fold
dilutions) to 0.3333,
0.1111, 0.0371, 0.0123, and 0.0041 dilutions (i.e., to yield 3.333 mg/ml,
which was then
diluted 3-fold to yield 1.111 mg/ml, etc.).
The FRTL-5 value for IgG sample #6 was 2080, while the FRTL-5 value for IgG
sample #11 was 4453, and for IgG sample #16, the value was 830. The following
Table 6
lists the results for each of these samples. The correlation coefficients (r)
were 0.857 for
IgG sample #6, 0.858 for sample #11, and 0.995 for sample #16.
Table 6. Dose-Response (Dilution) Curves of Graves' IgG Specimens*
Sample Dilution Factor
1 0.3333 0.1111 0.0371 0.0123
0.0041
IgG #6 176,123 159,694 62,115 13,480 -6,628 -
2,574
IgG #11 Not Done 368,373 324,143 158,641 77,298
30,166
IgG #16 222,413 90,646 40,048 8,093 -1,705 -691
*All values are reported as RLU/sec.
EXAMPLE 7
Alternative Protocol Using PEG
In these experiments, alternative protocols using PEG were tested. First,
freezer
vials of CHO-Rluc cells were thawed, diluted in Growth Medium (the contents of
each
cell vial were added to 2.5 ml medium), and 100 IA of this cell suspension
were added to
each of the 24 gelatin-coated wells of a 96-well microtiter plate, prepared as
described
previously. The plates were incubated for 20-24 hours in a 35- 37 C,
humidified
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incubator with an atmosphere containing 5% CO2. This provided monolayers that
were
loosely confluent.
The Growth Medium was removed and the monolayers rinsed with 100 p.1 of
Starvation Medium (normal HBSS with Ca ++ and Mg), and a final 100 I were
added to
each monolayer before incubating overnight under the conditions described
above.
Following incubation, the Starvation Medium was removed and 100 pl of
Stimulation
Medium containing 6% PEG (i.e., as described above) were added to each
monolayer.
Then, 10 pl of each of the standards and samples were placed into the wells
(in
triplicate). While other volumes were tested (e.g., 25 1, 50 p 1, and 75 IA),
the values
obtained were substantially equivalent to those obtained with 10 p.I volumes.
Thus, the
smaller volume was used in order to conserve the samples and reagents, and to
minimize
the concentration of potentially interfering substances present in some serum
samples.
The well contents were mixed and the monolayers incubated as described above
for 4 hours (i.e., a stimulation step). The medium was removed from each well,
and 150
IA of lysis solution (as described above) were added to each well. The
monolayers were
allowed to stand at room temperature for 30 minutes for lysis to occur. Then,
25 1 of
each lysate were added to individual luminometer tubes. Fifty microliters of
luciferase
substrate (as described above) were added to each tube, the contents mixed,
and the tubes
immediately read in a luminometer with settings of 5 seconds delay and a 10
second read
time.
In an experiment to determine the normal range of euthyroid sera, 24 specimens
obtained from a reference laboratory were run using the CHO-Rluc assay as
described
above. The sera were euthyroid in that none of the samples were submitted for
thyroid
testing. The mean (55,334 RLU/sec) and standard deviation (1 SD 7,434 RLU/sec)
were
calculated for these 24 euthyroid samples. The results are shown in Figure 7.
The SD
value was then multiplied by three, which yielded a cut-off for normal, non-
Graves'
disease values of 77,636 RLU/sec. This cut-off encompasses >99% of the normal
population; values greater than this were considered to be TSI positive.
In a separate set of experiments, a group of 17 patient specimens which
previously been tested by a commercial esoteric testing laboratory using cAMP
RIA and
FRTL-5 cells for TSI, were tested using the CHO-Rluc cells with the above
procedure.
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The FRTL-5 test results indicated 16 of the patient specimens were negative
for TSI (i.e.,
only one was positive). The single positive specimen identified by the FRTL-
5/cAMP
assay (258% or 1.98x the cut-off, where the assay cut-off was 130%), was
likewise
positive by the CHO-Rluc assay (190,691 RLU/sec) based on a 2.45x cut-off of
77,636
RLU/sec, as shown in Figure 7. The CHO-Rluc values of the 16 patient specimens
which
were negative (i.e., normal) by the FRTL-5/cAMP assay were found to be in good
agreement with the 24 normal sera used to establish the normal range for the
assay. See,
Figure 7.
EXAMPLE 8
Comparison of CHO-Rluc Method and Standard Methods
In these experiments, the methods of the present invention utilizing
Stimulation
Medium containing 6% PEG-8000 were compared with methods using the standard
HBSS-containing Starvation Medium and Stimulation Medium, to obtain luciferase
values for 35 of the untreated Graves' disease IgG specimens obtained from Dr.
Cho. The
cAMP values obtained by Dr. Cho with FRTL-5 and CHO-R cells using the same IgG
samples as used in methods of the present invention are shown in comparison
with the
CHO-R luciferase results in Figures 2, 3 and 4. Figure 5 shows the linearity
of luciferase
response to bTSH.
Figure 2 provides a comparison of CHO-Rluc luciferase results with the FRTL-5
cAMP results. This Figure indicates that the correlation between these methods
is quite
good. Figure 3 provides a comparison of CHO-Rluc luciferase results with CHO-R
cAMP results. The CHO-R CAMP cut-off value was 173. Values below this cutoff
were
as follows (CHOluc RLU/sec): 110 (219,913), 113 (14,434), 116 (25,373), 152
(84,493),
156 (7576), and 161 (61,321). As indicated in this Figure, the range of CHO-R
cAMP
results is relatively narrow, as compared with the CHO-Rluc values. This is
also shown in
Figure 4, which.provides a comparison of CHO-R cAMP results with FRTL-5 cAMP
results. The CHO-R value was 173. The FRTL-5 cut-off value was 153. Values
below
cutoff were as follows (FRTL-5 values): 110 (830), 113 (283), 116 (212), 152
(1100),
156 (388), and 161 (335). The average +/- SD values for the IgG Control (ICN),
for the
tests shown in Figure 2 were 472+/-4015 (n=8).
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Figure 5 shows the linearity of the response to bTSH of the CHO-Rluc cells. In
these experiments, dilutions of bTSH were tested. The RLU/sec values obtained
are
shown in Table 7, below.
Table 7. Results for bTSH Dilutions
Results j.tIU TSH.m1
0 10 25 50 75 100
RLU/sec 0 5,921 20,227 34,426 54,396
62,206
It is contemplated that this linearity and sensitivity of response to bTSH
will
prove useful in the detection of blocking antibodies to the TSH receptor
(e.g., those
autoantibodies in patents with atrophic thyroiditis and Hashimoto's
thyroiditis which
block the TSH receptor, thereby preventing thyroid hormone production and
release
resulting in hypothyroidism). This Figure also provides at least a partial
explanation of
why the CHO-R cell line is not as sensitive to TSI from Graves' disease
patients sera as
the FRTL-5 cell line. In these results, the correlation coefficient (r) was
0.9925. The three
S.D. (standard deviations) sensitivity was 1.3 IU TSH/ml.
EXAMPLE 9
Monitoring of Immune Responses
In these experiments, the immune response of vaccine recipients is measured
and
monitored. Although it is not intended that the present invention be so
limited, this
Example describes the monitoring of a subject's immune response to herpes
simplex
(HSV) vaccine.
Prior to administration of vaccine, a serum sample (i.e., preimmune serum) is
collected from the subject for use as a baseline or control, and stored frozen
until testing.
Serum samples are also collected at periodic intervals following
administration of the
vaccine (e.g., 1-2 weeks, 1 month, 2 months post-vaccination, etc.). The sera
are thawed
as necessary, and used in an assay to determine the presence and quantity
(i.e., titer) of
neutralizing antibodies. Sera are serially diluted and mixed with known
quantities of
HSV. These samples are diluted in dilutent comprising Eagle's MEM with HBSS
containing 2 mM glutamine, 2% FBS, and PEG (e.g., 6% PEG 8000). However, it is
also
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contemplated that other diluents will find use in the present method,
including diluents
containing different concentrations and types of PEG, as appropriate for the
virus and
assay system used). These samples are added to cell monolayers containing
cells capable
of producing an enzyme such as (3-galactosidase upon infection with HSV (e.g.,
ELVISTM cells, Diagnostic Hybrids). Following overnight incubation under
standard cell
culture conditions, the monolayers are lysed and the enzyme activity is
measured using
chromogenic or luminogenic methods.
A positive response to the vaccine is indicated by the lowest dilution of
postvaccination serum which neutralizes HSV in the sample (i.e., as indicated
by a low
OD. or luminescence value, in comparison with the preinunune control).
In summary, the present invention provides numerous advances and advantages
over the prior art, including the avoidance of radioactivity, in combination
with the
advantages of ease of use, reliability, sensitivity, specificity, cost-
effectiveness, and
reproducibility.
EXAMPLE 10
Construction Chimeric TSH-R Plasmids
This example presents one embodiment of constructing a cell line comprising a
chimeric TSH-R receptor for detecting Graves' disease autoantibodies.
Plasmid Construction
A plasmid comprising a first nucleic acid sequences encoding a TSH-R chimeric
receptor and a second nucleic acid sequence encoding a neomycin resistant gene
was
ligated to a luciferase gene and a glycoprotein hormone alpha subunit
promoter.
Human glycoprotein alpha subunit promoter cloning
Chromosomal DNA was isolated from human embryonic kidney cells using a
QIAGEN RNA/DNA kit (QIAGEN Cat#14123.) Glycoprotein alpha subunit promoter
fragments were amplified by PCR using the isolated chromosomal DNA as the PCR
template
and the 2 pairs of oligo-nucleotide primers shown below:
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5'PCR primer: 5'-GAGCTC ATG TGT ATG GCT CAA TAA AAT TAC GTA CAA
AGT GAC AGC -3'
3' PCR primer: 5'- AGATCT TCG TCT TAT GAG TTC TCA GTA ACT GCA GTA
TAA TGA AGT -3'.
A Sac I restriction site was added to the 5' end of the 5' PCR primer while a
Bgl
II restriction site was added to the 5' end of the 3' PCR primer (both shown
as underlined
sequence). For PCR amplification, BD Advantage 2 Polymerase Mix (BD Bioscience
Palo Alto CA) was used and PCR reactions were performed in a thermal cycler
(Eppendorf Mastercycler Personal, Germen.). Forty cycles were carried out at
94 C for
30 seconds to denature the DNA. Samples were then annealed to the primers in
the
thermalcycler at 63 C for 30 seconds, and the extension was induced at 68 C
for 1
minute 30 seconds per cycle. Two amplicons (1.2 kb and 0.6 kb) were cloned
into the
plasmid vector pcDNA2.1 (Invitrogen, Carlsbad, CA) and sequenced using the
BigDye
Terminator v3.0 Cycle Sequencing method on an ABI 377 automated sequencer
(Davis
Sequencing Inc.).
Construction of plasmid pGHP/Luc
The human glycoprotein alpha subunit promoter was isolated from vector
pcDNA2.1 by restriction cleavage with Sac I and Bgl II. The resulting 316 bp
fragment
was then subcloned into the Sac I/Bgl II site of the pGL2 enhancer plasmid
(Promega,
Madison, WI) for construction of a plasmid named pGHP/Luc.
Construction of plasmid pMc4-neo
The neomycin resistance gene for antibiotic selection (positive clone
selection)
was isolated from vector pMC 1 (Stratagene Cedar Creek, TX) with restriction
enzymes
of 'Choi and Hid!. The resulting fragment was then subcloned into the XbaI
site of
plasmid pMc4 that contains the TSHR/LH chimeric receptor driven by the SV40
promoter (from Dr. Leonard Kohn.) The final plasmid was named pMc4-neo.
Construction of plasmid pMc4-Bsd
The antibiotic selection gene Blastocidin, isolated from vector pCMV/Bsd
(Invitrogen, Carlsbad, CA) with restriction enzymes XhoI and XbaI, was
subcloned into
the XbaI site of plasmid pMc4 which contains the TSHR/LH chimeric receptor.
Tahara et
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al., "Immunoglobulins From Graves' Disease Patients Interact With Different
Sites On
TSH Receptor/LH/CG Receptor Chimeras Than Either TSH Or Immunoglobulins From
Idiopathic Myxedema Patients" Biochem Biophys Res Comm 179:70-77 (1991). The
final plasmid was named pMc4-Bsd.
Construction of plasmid pMc4-GHP/Luc
The human glycoprotein alpha subunit promoter, with a firefly luciferase
reporter
gene, was isolated from vector pMc4/Luc followingrestriction cleavage by SmaI
and
AccI. The isolated DNA fragment was then subcloned into the PfoI site of pMc4-
neo
plasmid. The final plasmid was named pMV4-GHP/Luc.
Example 11
Mammalian Cell Selection
Seven different mammalian cell lines were tested to select the cell line that
had
the lowest cyclic AMP basal level and highest potential inducible levels. The
results
demonstrated that the CHO and RD cells showed the lowest cyclic AMP basal
activity
and the highest potential inducible level. This empirical research approach
maximizes
the assay sensitivity by proper selection of cell culture type. For example, a
lower cyclic
AMP basal level increases the sensitivy of the luciferase assay. Also, the
highest induced
expression of cyclic AMP improves the accuracy of the luciferase assay.
Example 12
Transfection/Selection Of A CHO Cell Line With Chimeric TSH-R Plasmid
This example describes the permanent transfection of CHO cells.
Chinese Hamster Ovary cell line (CHO-Kl; ATCC Number: CCL-61, Manassas VA)
was transfected with a linearized (Xmnl) pMc4-GPH/Luciferase plasmid using
HyFect
(Denville Scientific, Metuchen, NJ.) according to the manufacturer's
instructions. The
CHO-Kl cells were then grown in Ham's F12 Medium with 10% (v/v) fetal bovine
serum and nine essential amino acids at 37 C in a humidified atmosphere
containing 5%
CO2. Twenty four hours after the transfection, the cells were combined and
planted into
a 96 well plate and selected with 0.5 mg/ml G418 in Ham's F12 Medium with 10%
FBS.
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Example 13
Transfection/Selection Of An RD Cell Line With Chimeric TSH-R Plasmid
This example describes the transfection of a Human Rhabdomyosarcoma (RD)
(ATCC Number: CCL-136.) cell line with two plasmids, pGHP/Luc and pMc4-Bsd, in
series to facilitate detection.
RD cells were transfected with the linearized pGPH/Luc (Seal) plasmid using
HyFect (Denville Scientific, Metuchen, NJ.) according to the manufacturer's
instructions.
The cells were selected with 0.5 mg/ml of neomycin. The optimal clone from
this
transfection and selection was then transfected with the linearized plasmid
pMc4-Bsd.
After transfection, the cells were selected with both neomycin (0.5mg/m1) and
blasticidin
(51.1g/m1.) to produce the final RD recombinant cell line.
All CHO and RD antibiotic resistant clones were tested with TSI-positive and
normal serum to select the clone which can be used for the detection of TSI.
The TSI
induction positive clone was subjected to the limiting dilution cloning to
further select a
single clone.
The final clones have the ability to diagnose Graves' disease and/or monitor
the
drug treatment of patients with Graves' disease with higher sensitivity than
the current
product on the market. These cell lines show good stability, having been
passaged more
than ten times, and continue to show very similar performance characteristics.
Example 14
Induction Of Cell Lines With TSI Containing Serum
CHO cells from freezer vials were diluted and grown in growth media (Ham's F12
Medium with 10 (v/v) % fetal bovine serum and nine essential amino acids) for
16 hours
at 37 C and 5% CO2. After 16 hours the media was removed and the CHO cells
were
rinsed and refed with 100 IA/ well "starvation" HBSS medium. The CHO cells
were then
incubated for 22-24 hours. Following incubation the media was removed and CHO
cells
were rinsed and refed with 100 ial/ well reaction buffer. The CHO cells were
then
induced with a 1:11 dilution of patient serum in reaction buffer containing
BSA, PEG,
sucrose, glucose, and salts (Diagnostic Hybrids Catalog number 40-300500;) for
4 hours
at 37 C and 5% CO2.
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RD cells were grown in Eagles Minimal Essential Medium (EMEM) with 10(v/v) %
fetal bovine serums at 37 C and 5% CO2 for 16 -24 hours. RD cells were then
directly
induced with patient serum in reaction buffer (Diagnostic Hybrids Catalog
number 40-
300500) for 4 hours at 37 C and 5% CO2.
All publications and patents mentioned in the above specification are herein
incorporated by reference. Various modifications and variations of the
described method
and system of the invention will be apparent to those skilled in the art
without departing
from the scope and spirit of the invention. Although the invention has been
described in
connection with specific preferred embodiments, it should be understood that
the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed,
various modifications of the described modes for carrying out the invention
which are
obvious to those skilled in diagnostics, cell culture, and/or related fields
are intended to
be within the scope of the following claims.
Example 15
Effect Of Dexamethasone On Starved CHO-RMc4luc Cells
CHO-Rluc (041307A) and CHO-RMc4luc (062707, P8, 3e6/m1) cells were each
brought up in 5 ml CHO growth media. From this stock of cells enough was taken
out
and aliquoted into 6 tubes so that there would be 66,666 cells/well for CHO-
Rluc assay
and 50,000 cells/well for the CHO-RMc4luc assay for each dexamethasone
concentration
(100, 50, 40, 25, 12.5, and 011M). The dexamethasone stock was 500 M in CHO-
growth
media. All dexamethasone was made fresh. These vials were spun down and the
cells
were brought up in the different concentrations of dexamethasone in CHO growth
media.
Cells were then grown for 24 hours, rinsed with starvation media, and then
incubated in dexamethasone-containing starvation media that contained the same
for 24
hours. The cells were then rinsed with reaction buffer and serum (diluted
1:11) was
added to 100 1 of reaction buffer already in the well. Serums used were: i)
reference
121506R; ii) PC 121506P; and iii) patient #18 TSI serum. Reference and Patient
#18
serums were both treated the same and incubated with all 6 concentrations of
dexamethasone.
63
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PCT/US2008/011027
After 4 hours of induction the plates were lysed with 75u1 of Bright Glo ,
lysed
for 5 minutes, and then read on a Veritas luminometer.
Example 16
Comparison Of Starvation Periods To Dexamethasone Treatment
Nine (9) human serum samples were prepared where four (4) samples were
known positive for Grave's Disease and five (5) samples were known negative
for
Grave's disease. Each of the nine samples was tested in each condition of both
the Non-
Starvation/Dexamethasone Protocol and the Starvation Protocol.
1. Non-Starvation/Dexamethasone Protocol
Three cell plates were analyzed using a CHO-RMc4 protocol. Plate 1: CHO-
Mc4luc cells (4e6 cells/plate) + Growth Medium containing 40 M dexamethasone
in
CHO-Mc4 Reaction Buffer. Plate 2: CHO-RMc4luc cells (4e6 cells/plate) + Growth
Medium without 40 M dexamethasone in CHO-Mc4 Reaction Buffer. Plate 3: CHO-
Rluc cells (4e6 cells/Plate) + Growth Medium without 40 tM dexamethasone in
CHO-
Rluc Reaction Buffer
Each plate underwent a 16 hour growth period, a 3 hour induction period, and a
10 minute lysis period.
2. Starvation Protocol
Three plates were analyzed using a CHO-R protocol. Plate 1: CHO-RMc4 cells
(4e6 cells/plate) + Growth Medium containing 40 1.1M dexamethasone in
CHO-Mc4 Reaction Buffer. Plate 2: CHO-RMc4luc cells (4e6 cells/plate) + Growth
Medium without 40 M dexamethasone in CHO-Mc4 Reaction Buffer. Plate 3: CHO-
Rluc cells (4e6 cells/Plate) + Growth Medium without 401.1M dexamethasone in
CHO-
Rluc Reaction Buffer. The sample arrangement for each protocol was configured
identically. See, Figure 16.
Each plate underwent a 24 hour growth period, a 24 hour starvation period, a 4
hour induction period, and a 5 minute lysis period. The luminosity for each
sample was
measured using a Veritas luminometer.
64
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PCT/US2008/011027
Example 17
Comparison Of Alternate Glucocortoids With Dexamethasone
This example provides data showing that improved sensitivity of a CHO-RMc4
assay is not limited to the substitution of a Starvation medium period with
dexamethasone (Dex) in accordance with Example 16. These data demonstrate that
four
alternative glucocorticoids (GCs) have equivalent effects in improving signal
intensity.
Four GCs were examined in this study: i) Prednisone (Sigma); ii)
Hydrocortisone
(Sigma); iii) Fluticasone Propionate (Sigma); and iv) Cortisone (Sigma).
A stock concentration (100 mM) of each GC was made in DMSO. 1:10 (10 mM) and
1:100 (1 mM) dilutions, in DMSO, were then made from the 100 mM stock. All
DMSO/GC stocks were clear with no visible precipitate.
Growth Media containing different concentrations of each GC were made as
follows: i) 100 M GC Medium: 5 L of 100 mM GC Stock + 5 mL SR097 (w/o Dex);
ii) 50 M GC Medium: 2.5 I, of 100 mM GC Stock +5 mL SR097 (w/o Dex); iii) 10
M GC Medium: 5 pL of 10 mM GC Stock + 5 mL SR097 (w/o Dex); iii) 1 M GC
Medium: 5 L of 1 mM GC Stock + 5mL SR097 (w/o Dex); and iv) 0.1 M GC Medium:
0.5 I, of 1 mM GC Stock + 5 mL SR097 (w/o Dex). A 40 M dexamethasone control
sample was run in comparison to these various concentrations of the
alternative
glucocorticoids. Additonal controls included Growth Medium containing 1 pl/m1
dimethylsulfoxide (DMSO) as a solvent control and Growth Medium without any
glucocorticoids.
Each assay was performed by using two (2) microwell plates for each
glucocorticoid tested. The sample layouts for each plate are identified below:
A. Plate One
A
L M N 0
A Positive (GM containing dexamethasone)
Reference (GM containing dexamethasone)
Normal (GM containing dexamethasone)
CA 02701198 2010-03-30
WO 2009/045292
PCT/US2008/011027
Positive (GM containing 100 M glucocorticoid)
Reference (GM containing 100 M glucocorticoid)
Normal (GM containing 100 M glucocorticoid)
Positive (GM containing 50 M glucocorticoid)
Reference (GM containing 50 M glucocorticoid)
Normal (GM containing 50 M glucocorticoid)
Positive (GM containing 10 M glucocorticoid)
Reference (GM containing 10 M glucocorticoid)
Normal (GM containing 10 M glucocorticoid)
Positive (GM containing 1 M glucocorticoid)
Normal (GM containing 1 M glucocorticoid)
0 Reference (GM containing 1 M glucocorticoid)
B. Plate Two
R S T U V
X Y Z AA
Positive (GM containing dexamethasone)
Reference (GM containing dexamethasone)
Normal (GM containing dexamethasone)
Positive (GM containing 0.1 M glucocorticoid)
Reference (GM containing 0.1 M glucocorticoid)
Normal (GM containing 0.1 p.M glucocorticoid)
V Positive (GM containing DMSO)
Reference (GM containing DMSO)
X Normal (GM containing DMSO)
Positive (GM only)
Reference (GM only)
AA Normal (GM only)
66
CA 02701198 2010-03-30
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The raw data is presented below. See Table 8.
Table 8: Alternative Glucocortiocids - Raw Data
,
Condition A (positive ¨ normal) A (positive ¨
normal)
RLU SRR%s
Fluticasone Proprionate
0.1 gIVI 9768 161
1 M 8806 148
!AM 8774 168
50 M 6996 137
100 M 6366 155
Prednisone
0.1 M 8717 150
1 M 8219 133
10 M 8429 142
50 M 8107 165
100 M Not Determined Not Determined
Hydrocortisone
0.1 M 9889 158
1 M 9149 155
10 M 9757 153
50 M 8950 178
100 M 8348 180
Cortisone
0.1 M 10494 169
1 M 9474 151
10 M 9847 151
50 M 8738 162
100 M 8106 176
67
CA 02701198 2010-03-30
WO 2009/045292
PCT/US2008/011027
Controls
40 AM Dexamethasone 9375 282
GM + 1 AL/m1 DMSO 9467 141
GM w/o Dexamethasone 9010 157
68
CA 02701198 2011-09-07
SEQUENCE LISTING IN ELECTRONIC FORM
This description contains a sequence listing in electronic form in ASCII
text format. A copy of the sequence listing in electronic form is
available from the Canadian Intellectual Property Office. The sequences in
the sequence listing in electronic form are reproduced in the following
Table.
SEQUENCE TABLE
<110> Diagnostic Hybrids, Inc.
<120> Sensitive and Rapid Methods of Using Chimeric Receptors to
Identify Autoimmune Disease
<130> 81344-130
<140> CA 2,701,198
<141> 2010-03-30
<150> 11/906,189
<151> 2007-10-01
<150> 12/206, 322
<151> 2008-09-08
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 1
gagctcatgt gtatggctca ataaaattac gtacaaagtg acagc 45
<210> 2
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 2
agatcttcgt cttatgagtt ctcagtaact gcagtataat gaagt 45
68a
CA 02701198 2011-09-07
_
<210> 3
<211> 2324
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 3
ggcgatttcg gaggatggag aaatagcccc gagtcccgtg gaaaatgagg ccggcggact
60
tgctgcagct ggtgctgctg ctcgacctgc ccagggacct gggcggaatg gggtgttcgt
120
ctccaccctg cgagtgccat caggaggagg acttcagagt cacctgcaag gatattcaac
180
gcatccccag cttaccgccc agtacgcaga ctctgaagct tattgagact cacctgagaa
240
ctattccaag tcatgcattt tctaatctgc ccaatatttc cagaatctac gtatctatag
300
atgtgactct gcagcagctg gaatcacact ccttctacaa tttgagtaaa gtgactcaca
360
tagaaattcg gaataccagg aacttaactt acatagaccc tgatgccctc aaagagctcc
420
ccctcctaaa gttccttggc attttcaaca ctggacttaa aatgttccct gacctgacca
480
aagtttattc cactgatata ttctttatac ttgaaattac agacaaccct tacatgacgt
540
caatccctgt gaatgctttt cagggactat gcaatgaaac cttgacactg aagctgtaca
600
acaatggctt tacttcagtc caaggatatg ctttcaatgg gacaaagctg gatgctgttt
660
acctaaacaa gaataaatac ctgacagtta ttgacaaaga tgcatttgga ggagtataca
720
gtggaccaag cttgctggac gtgtctcaaa ccagtgtcac tgcccttcca tccaaaggcc
780
tggagcacct gaaggaactg atagcaagaa acacctggac tcttaagaca ctgccctcca
840
aagaaaaatt cacgagcctc ctggtcgcca cgctgaccta ccccagccac tgctgcgcct
900
tcagtaattt gccgaagaaa gaacagaatt tttcattttc catttttgaa aacttctcca
960
aacaatgcga aagcacagtt agaaaagcag ataacgagac gctttattcc gccatctttg
1020
aggagaatga actcagtggc tgggatgagc tcaaaaaccc ccaggaagag actctacaag
1080
cttttgacag ccattatgac tacaccatat gtggggacag tgaagacatg gtgtgtaccc
1140
ccaagtccga tgagttcaac ccgtgtgaag acataatggg ctacaagttc ctgagaattg
1200
tggtgtggtt cgttagtctg ctggctctcc tgggcaatgt ctttgtcctg cttattctcc
1260
tcaccagcca ctacaaactg aacgtccccc gctttctcat gtgcaacctg gcctttgcgg
1320
atttctgcat ggggatgtac ctgctcctca tcgcctctgt agacctctac actcactctg
1380
agtactacaa ccatgccatc gactggcaga caggccctgg gtgcaacacg gctggtttct
1440
tcactgtctt tgcaagcgag ttatcggtgt atacgctgac ggtcatcacc ctggagcgct
1500
ggtatgccat caccttcgcc atgcgcctgg accggaagat ccgcctcagg cacgcatgtg
1560
68b
CA 02701198 2011-09-07
ccatcatggt tgggggctgg gtttgctgct tccttctcgc cctgcttcct ttggtgggaa 1620
taagtagcta tgccaaagtc agtatctgcc tgcccatgga caccgagacc cctcttgctc 1680
tggcatatat tgtttttgtt ctgacgctca acatagttgc cttcgtcatc gtctgctgct 1740
gttatgtgaa gatctacatc acagtccgaa atccgcagta caacccaggg gacaaagata 1800
ccaaaattgc caagaggatg gctgtgttga tcttcaccga cttcatatgc atggccccaa 1860
tctcattcta tgctctgtca gcaattctga acaagcctct catcactgtt agcaactcca 1920
aaatcttgct ggtactcttc tatccactta actcctgtgc caatccattc ctctatgcta 1980
ttttcaccaa ggccttccag agggatgtgt tcatcctact cagcaagttt ggcatctgta 2040
aacgccaggc tcaggcatac cgggggcaga gggttcctcc aaagaacagc actgatattc 2100
aggttcaaaa ggttacccac gacatgaggc agggtctcca caacatggaa gatgtctatg 2160
aactgattga aaactcccat ctaaccccaa agaagcaagg ccaaatctca gaagagtata 2220
tgcaaacggt tttgtaagtt aacactacac tactcacaat ggtaggggaa cttacaaaat 2280
aatagtttct tgaatatgca ttccaatccc atgacacccc caac 2324
<210> 4
<211> 304
<212> DNA
<213> Homo sapiens
<400> 4
atgtgtatgg ctcaataaaa ttacgtacaa agtgacagcg tactctcttt tcatgggctg 60
accttgtcgt caccatcacc tgaaaatggc tccaaacaaa aatgacctaa gggttgaaac 120
aagataagat caaattgacg tcatggtaaa aattgacgtc atggtaatta caccaagtac 180
ccttcaatca ttggatggaa tttcctgttg atcccagggc ttagatgcag gtggaaacac 240
tctgctggta taaaagcagg tgacgacttc attatactgc agttactgag aactcataag 300
acga 304
<210> 5
<211> 337
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic
<400> 5
attcgccctt gagctcatgt gtatggctca ataaaattac gtacaaagtg acagcgtact 60
ctcttttcat gggctgacct tgtcgtcacc atcacctgaa aatggctcca aacaaaaatg 120
68c
CA 02701198 2011-09-07
. acctaagggt tgaaacaaga taagatcaaa ttgacgtcat ggtaaaaatt gacgtcatgg
180
taattacacc aagtaccctt caatcattgg atggaatttc ctgttgatcc cagggcttag
240
atgcaggtgg aaacactctg ctggtataaa agcaggtgag gacttcatta tactgcagtt
300
actgagaact cataagacga agatctaagg gcgaatt
337
<210> 6
<211> 85
<212> PRT
<213> Bos taurus
<400> 6
Gin Thr Leu Ile Ala Thr Ser Ser Tyr Ser Leu Lys Lys Leu Pro Ser
1 5 10 15
Arg Glu Lys Phe Ala Asn Leu Leu Asp Ala Thr Leu Thr Tyr Pro Ser
20 25 30
His Cys Cys Ala Phe Arg Asn Val Pro Thr Lys Glu Gin Asn Phe Her
35 40 45
Phe Ser Ile Ser Lys Asn Phe Pro Lys Gin Cys Glu Ser Thr Val Arg
50 55 60
Lys Gin Asn Asn Glu Thr Leu Tyr Pro Ala Ile Phe Ala Glu Ser Gly
65 70 75 80
Gin Ser Gly Trp Asp
<210> 7
<211> 173
<212> PRT
<213> Bos taurus
<400> 7
Ile Leu Ile Leu Asn Thr Lys Asn Leu Leu His Ile Glu Asp Gly Ala
1 5 10 15
Phe Arg Asn Leu Pro Arg Leu Lys Tyr Leu Ser Ile Cys Asn Thr Gly
20 25 30
Ile Ile Glu Phe Pro Asp Leu Thr Gin Ile Phe Ser Ser Glu Ala His
35 40 45
68d
CA 02701198 2011-09-07
Phe Ile Leu Glu Leu Cys Asp Asn Leu Arg Met Thr Thr Ile Pro Gin
50 55 60
Asn Ala Phe Arg Gly Met Ser Asn Glu Ser Leu Thr Leu Lys Leu Tyr
65 70 75 80
Lys Asn Gly Phe Glu Asp Ile His Ser His Ala Phe Asn Gly Thr Lys
85 90 95
Leu Asn Gin Leu Ile Leu Lys Asp Asn Lys Asn Leu Arg Arg Ile His
100 105 110
Asn Asp Ala Leu Arg Gly Ala Ile Gly Pro Asp Val Leu Asp Ile Ser
115 120 125
Ser Thr Ala Leu Glu Ser Leu Pro Ser Tyr Gly Leu Glu Ala Ile Gin
130 135 140
Val Leu Asn Gly Met Ser Ser Tyr Ser Leu Lys Arg Leu Pro Pro Leu
145 150 155 160
Asp Lys Phe Ser Ser Leu Leu Glu Ala Val Leu Thr Tyr
165 170
<210> 8
<211> 728
<212> PRT
<213> Bos taurus
<400> 8
Met Leu Pro Ala Leu Leu Pro Leu Leu Leu Pro Ala Leu Leu Pro Gly
1 5 10 15
Ala Gly Gly Gly Arg Cys Pro Gin Arg Cys Ala Cys Thr Gin Pro Ala
20 25 30
Leu Arg Cys Pro Thr Pro Pro Pro Gly Ala Arg Pro Ala Pro Ala Arg
35 40 45
Ala Ser Phe Thr His Leu Pro Val Lys Val Ile Pro Ser His Ala Phe
50 55 60
Glu Gly Leu Arg Asp Ala Phe Ile Ile Glu Ile Ser Gln Ser Asp Ser
65 70 75 80
68e
CA 02701198 2011-09-07
Leu Glu Arg Ile Glu Ala Ser Ala Phe Asp Ser Leu Pro Ala Leu Ser
85 90 95
Glu Ile Leu Ile Leu Asn Thr Lys Asn Leu Leu His Ile Glu Asp Gly
100 105 110
Ala Phe Arg Asn Leu Pro Arg Leu Lys Tyr Leu Ser Ile Cys Asn Thr
115 120 125
Gly Ile Ile Glu Phe Pro Asp Leu Thr Gin Ile Phe Ser Ser Glu Ala
130 135 140
His Phe Ile Leu Glu Leu Cys Asp Asn Leu Arg Met Thr Thr Ile Pro
145 150 155 160
Gin Asn Ala Phe Gin Gly Met Ser Asn Glu Ser Leu Thr Leu Lys Leu
165 170 175
Tyr Lys Asn Gly Phe Glu Asp Ile His Ser His Ala Phe Asn Gly Thr
180 185 190
Lys Leu Asn Gin Leu Ile Leu Lys Asp Asn Lys Asn Leu Arg Arg Ile
195 200 205
His Asn Asp Ala Leu Arg Gly Ala Thr Gly Pro Asp Val Leu Asp Ile
210 215 220
Ser Ser Thr Ala Leu Glu Ser Leu Pro Ser Tyr Gly Leu Glu Ala Ile
225 230 235 240
Gin Val Leu Asn Ala Met Ser Ser Tyr Ser Leu Lys Arg Leu Pro Pro
245 250 255
Leu Asp Lys Phe Ser Ser Leu Leu Glu Ala Val Leu Thr Tyr Pro Ser
260 265 270
His Cys Cys Ala Phe Gin Asn Leu Arg Thr Glu Lys Gin Asn Ser Leu
275 280 285
Leu Ser Ile Phe Asp Asn Phe Ser Lys Gin Cys Glu Ser Thr Met Arg
290 295 300
Lys Pro Ala Ser Glu Val Phe Tyr Arg Asp Ala Ser Ser Asn Thr Ser
305 310 315 320
68f
CA 02701198 2011-09-07
Leu Trp Pro Ala Glu Lys His Met Tyr Pro Leu Glu Thr Gly Glu Glu
325 330 335
Ala Phe Pro Tyr Ser Tyr Ser Thr Val Phe Tyr Glu Asp Glu Met Thr
340 345 350
Gly Phe Asp Phe Glu Tyr Asp Phe Cys Gln Pro Lys Ile Leu Thr Cys
355 360 365
Thr Pro Glu Pro Asp Ala Phe Asn Pro Cys Glu Asp Ile Leu Gly Tyr
370 375 380
Ser Phe Leu Arg Val Leu Ile Trp Phe Ile Asn Ile Leu Ala Leu Ala
385 390 395 400
Gly Asn Phe Ile Val Leu Leu Val Leu Ile Thr Ser His Tyr Lys Leu
405 410 415
Thr Val Pro Arg Phe Leu Met Cys Asn Leu Ser Phe Ala Asp Phe Cys
420 425 430
Met Gly Leu Tyr Leu Leu Leu Ile Ala Ser Val Asp Ala Gln Thr Ser
435 440 445
Gly Gln Tyr Tyr Asn His Ala Ile Asp Trp Gin Thr Gly Ser Gly Cys
450 455 460
Ser Thr Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr
465 470 475 480
Thr Leu Thr Val Ile Thr Ile Glu Arg Trp His Thr Ile Thr Tyr Ala
485 490 495
Met Gln Leu Asp Arg Lys Leu Arg Leu Arg His Ala Val Pro Ile Met
500 505 510
Leu Gly Gly Trp Val Phe Ser Ile Leu Ile Ala Val Leu Pro Leu Leu
515 520 525
Gly Val Ser Ser Tyr Met Lys Val Ser Ile Cys Leu Pro Met Asp Ile
530 535 540
Glu Thr Gly Leu Ser Gln Ala Tyr Ile Leu Leu Ile Leu Met Leu Asn
545 550 555 560
68g
CA 02701198 2011-09-07
Val Ile Ala Phe Leu Val Ile Cys Ala Cys Tyr Ile Lys Ile Tyr Val
565 570 575
Ala Val Gin Asn Pro Glu Leu Val Ala Ala Asn Lys Asp Thr Lys Ile
580 585 590
Ala Lys Arg Met Ala Ile Leu Ile Phe Thr Asp Phe Thr Cys Met Ala
595 600 605
Pro Ile Ser Phe Phe Ala Ile Ser Ala Ala Ile Lys Val Pro Leu Ile
610 615 620
Thr Val Thr Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Val Asn
625 630 635 640
Ser Cys Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gin
645 650 655
Arg Asp Phe Phe Leu Leu Met Ser Lys Leu Gly Cys Cys Lys Ser Arg
660 665 670
Ala Glu Leu Tyr Arg Val Asn Tyr Phe Ser Ala Tyr Thr Pro Asn Cys
675 680 685
Lys Asn Gly Ser Ser Ala Pro Gly Pro Ser Lys Ala Ser Gin Ala Leu
690 695 700
Leu Leu Leu Ser Ala Ser Glu Lys Leu Cys Lys Thr Arg Arg Ser Thr
705 710 715 720
Lys Lys Ser Gin Pro Glu Cys Gin
725
<210> 9
<211> 58
<212> PRT
<213> Bos taurus
<400> 9
Met Gly Arg Arg Val Pro Ala Leu Arg Gin Leu Leu Val Leu Ala Met
1 5 10 15
Leu Val Leu Lys Gin Ser Gin Leu His Ser Pro Glu Leu Ser Gly Ser
20 25 30
68h
CA 02701198 2011-09-07
Arg Cys Pro Glu Pro Cys Asp Cys Ala Pro Asp Gly Ala Leu Arg Cys
35 40 45
Pro Gly Pro Arg Ala Gly Leu Ala Arg Leu
50 55
<210> 10
<211> 701
<212> PRT
<213> Bos taurus
<400> 10
Met Gly Arg Pro Ser Leu Ala Leu Arg Leu Leu Leu Ala Leu Leu Leu
1 5 10 15
Leu Pro Pro Pro Ala Pro Leu Leu Trp Ala Leu Arg Pro Ala Pro Cys
20 25 30
Pro Glu Pro Cys Ser Cys Pro Pro Asp Gly Ala Leu Arg Cys Pro Gly
33 40 45
Pro Gin Ala Gly Leu Ser Arg Leu Ser Leu Thr Tyr Leu Pro Ile Lys
50 55 60
Val Ile Pro Ser Gin Ala Phe Arg Gly Leu Asn Glu Val Ile Lys Ile
65 70 75 80
Glu Ile Ser Gin Ser Asp Ser Leu Glu Lys Ile Glu Ala Asn Ala Phe
85 90 95
Asp Asn Leu Leu Asn Leu Ser Glu Ile Leu Ile Gin Asn Thr Lys Asn
100 105 110
Leu Val His Ile Glu Ala Gly Ala Phe Thr Asn Leu Pro Arg Leu Lys
115 120 125
Tyr Leu Ser Ile Cys Asn Thr Gly Ile His Lys Leu Pro Asp Val Thr
130 135 140
Lys Ile Phe Ser Ser Glu Phe Asn Phe Ile Leu Glu Ile Cys Asp Asn
145 150 155 160
Leu His Ile Thr Thr Ile Pro Arg Asn Ala Phe Gin Gly Met Asn Asn
165 170 175
68i
CA 02701198 2011-09-07
Glu Ser Ile Thr Leu Lys Leu Tyr Gly Asn Gly Phe Glu Glu Ile Gin
180 185 190
Ser His Ala Phe Asn Gly Thr Thr Leu Ile Ser Leu Glu Leu Lys Glu
195 200 205
Asn Ala Arg Leu Glu Lys Met His Asn Asp Ala Phe Arg Gly Ala Thr
210 215 220
Gly Pro Ser Ile Leu Asp Ile Ser Ser Thr Lys Leu Gin Ala Leu Pro
225 230 235 240
Thr Tyr Gly Leu Glu Ser Ile Gin Thr Leu Ile Ala Thr Ser Ser Tyr
245 250 255
Ser Leu Lys Lys Leu Pro Ser Arg Glu Lys Phe Thr Asn Leu Leu Asp
260 265 270
Ala Thr Leu Thr Tyr Pro Ser His Cys Cys Ala Phe Arg Asn Leu Pro
275 280 285
Thr Asn Glu Gln Asn Phe Ser Phe Ser Ile Phe Lys Asn She Ser Lys
290 295 300
Gin Cys Glu Ser Thr Ala Arg Arg Pro Asn Asn Glu Thr Leu Tyr Ser
305 310 315 320
Ala Ile Phe Ala Glu Ser Glu Leu Ser Gly Trp Asp Tyr Asp Tyr Gly
325 330 335
She Cys Leu Pro Lys Thr Leu Gin Cys Ala Pro Glu Pro Asp Ala Phe
340 345 350
Asn Pro Cys Glu Asp Ile Met Gly Tyr Asn She Leu Arg Val Leu Ile
355 360 365
Trp Leu Ile Asn Ile Leu Ala Ile Thr Gly Asn Val Thr Val Leu She
370 375 380
Val Leu Leu Thr Ser Arg Tyr Lys Leu Thr Val Pro Arg Phe Leu Met
385 390 395 400
Cys Asn Leu Ser Phe Ala Asp Phe Cys Met Gly Leu Tyr Leu Leu Leu
405 410 415
68j
CA 02701198 2011-09-07
Ile Ala Ser Val Asp Ala Gin Thr Lys Gly Gin Tyr Tyr Asn His Ala
420 425 430
Ile Asp Trp Gin Thr Gly Ser Gly Cys Ser Ala Ala Gly Phe Phe Thr
435 440 445
Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu Thr Val Ile Thr Leu
450 455 460
Glu Arg Trp His Thr Ile Thr Tyr Ala Ile Gin Leu Asp Gin Lys Leu
465 470 475 480
Arg Leu Lys His Ala Ile Pro Val Met Leu Gly Gly Trp Leu Phe Ser
485 490 495
Thr Leu Ile Ala Val Leu Pro Leu Val Gly Val Ser Asn Tyr Met Lys
500 505 510
Val Ser Ile Cys Leu Pro Met Asp Val Glu Ser Thr Leu Ser Gin Val
515 520 525
Tyr Ile Leu Thr Ile Leu Ile Leu Asn Val Met Ala Phe Ile Ile Ile
530 535 540
Cys Ala Cys Tyr Ile Lys Ile Tyr Phe Ala Val Gin Asn Pro Glu Leu
545 550 555 560
Met Ala Thr Asn Lys Asp Thr Lys Ile Ala Lys Lys Met Ala Val Leu
565 570 575
Ile Phe Thr Asp Phe Thr Cys Met Ala Pro Ile Ser Phe Phe Ala Ile
580 585 590
Ser Ala Ala Phe Lys Val Pro Leu Ile Thr Val Thr Asn Ser Lys Val
595 600 605
Leu Leu Val Leu Phe Tyr Pro Val Asn Ser Cys Ala Asn Pro Phe Leu
610 615 620
Tyr Ala Ile Phe Thr Lys Ala Phe Gin Arg Asp Phe Phe Leu Leu Leu
625 630 635 640
Ser Lys Phe Gly Cys Cys Lys Tyr Arg Ala Glu Leu Tyr Arg Arg Lys
645 650 655
68k
CA 02701198 2011-09-07
= Asp Phe Ser Ala Tyr Ile Ser Asn Cys Lys Asn Gly Phe Thr Gly Ser
660 665 670
Asn Lys Pro Ser Arg Ser Thr Phe Lys Leu Thr Thr Leu Gin Cys Gin
675 680 685
Tyr Ser Ala Val Leu Asp Lys Thr Cys Tyr Lys Glu Cys
690 695 700
=
681
