Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-1-
METHOD TO DETECT BIOLOGICALLY ACTIVE, ALLERGEN-SPECIFIC
IMMUNOGLOBULINS
FIELD OF THE INVENTION
The present invention relates to a novel method to detect biologically active,
allergen-specific immunoglobulins. The present invention also includes novel
kits to
detect such immunoglobulins. The present invention also includes novel
immunoglobulins.
BACKGROUND OF THE INVENTION
Diagnosis of disease and determination of treatment efficacy are important
tools
in medicine. In particular, detection of immunoglobulin E (IgE) production in
an animal
can be indicative of disease. Such diseases include, for example, allergy,
asthma, atopic
disease, hyper IgE syndrome, internal parasite infections and B cell
neoplasia. IgE-
mediated allergic diseases, such as asthma, hay fever, atopic dermatitis and
other skin
diseases, affect up to 30 percent of the human population of industrialized
countries. In
addition, reduction of IgE production in an animal following a treatment is
indicative of
the efficacy of the treatment, such as when using treatments intended to
disrupt IgE
production.
Until recently, the diagnosis of IgE-mediated disease and the identification
of the
responsible allergens have been based primarily on a patient's history, on
skin testing
and on serological in vitro detection of IgE using antibodies that bind
selectively to
epsilon isotype antibodies (i.e., anti-IgE antibodies). These anti-IgE
antibodies,
however, have several drawbacks: many anti-IgE antibodies cross-react with
other
antibody isotypes, such as gamma isotype antibodies, leading to assays having
Iow
specificity; results from tests using anti-IgE antibodies often do not
correlate with
biological test results, such as skin tests, provocation tests, or a patient's
history; anti-
IgE antibodies can be inhibited from binding to IgE when the IgE is bound to
its
respective allergen; anti-IgE antibodies cannot distinguish between
biologically active
and inactive IgE; and anti-IgE antibodies cannot identify other IgE-like
molecules that
interact with the high affinity receptor for IgE to cause allergy.
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
_2_
The high affinity receptor for IgE (FcERT) consists of three protein chains:
alpha,
beta and gamma. Prior investigators have disclosed the nucleic acid sequence
for: the
alpha chain (U.S. Patent No. 4,962,035, by Leder, issued October 9, 1990; U.S.
Patent
No. 5,639,660, by Kinet et al., issued June 17, 1997; Kochan et al., Nucleic
Acids Res.
16:3584, 1988; Shimizu et al., Proc. Natl. Acad. Sci. USA 85:1907-1911, 1988;
and
Pang et al., J. Immunol. 151:6166-6174, 1993}; the beta chain (Kuster et al.,
J. Biol.
Chem. 267:12782-12787, 1992); and the gamma chain {Kuster et al., J. Biol.
Chem.
265:6448-6452, 1990). These investigators, however, did not disclose the use
of such a
receptor, or subunits thereof, to detect IgE in animals, such as allergy-
mediating IgE.
There have been a few reports of the use of the human FcERI alpha chain to
detect total IgE in human sera, but to the inventors' knowledge, such reports
do not
present evidence of the ability to use the FcERI alpha chain to identify
allergen-specific
IgE; see, for example, 3apanese Patent Application Publication 8-101194, by
Ami,
published April 16, 1996; and Suto et al, Jpn. J. Dermatol. 106, 1377-1384,
1996. Not
demonstrating the ability of FcERI to identify allergen-specific IgE is
relevant because
there are teachings that binding of allergen to IgE inhibits the ability of
the IgE to bind
to the FcERI alpha chain and that, as such, FcERI might not be a good reagent
for the
detection of allergen-specific IgE ; see, for example, Stampfli et al, Journal
of
Immunology 155, 2948-2954, 1995.
Thus, methods and kits are needed in the art that will provide detection of
biologically active, allergen-specific immunoglobulins.
SUMMARY OF THE INVENTION
The present invention includes a method to detect a biologically active
immunoglobulin that selectively binds to a specific allergen in a mammal. The
method
includes the steps of (a) contacting a putative biologically active, allergen-
specific
immunoglobulin-containing composition from the mammal with an isolated FcE
receptor
(FcER) molecule and with the specific allergen under conditions suitable for
formation of
a FcER:immunoglobulin:allergen complex; and (b) determining the presence of
the
immunoglobulin by detecting the complex, the presence of the complex
indicating the
presence of the immunoglobulin.
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-3-
One embodiment of the present invention is a method to detect a biologically
active, allergen-specific immunoglobulin in a mammal, wherein a process using
anti-IgE
antibodies does not detect the immunoglobulin. The method includes the steps
of (a)
contacting a putative biologically active, allergen-specific immunoglobulin-
containing
composition from the mammal with an isolated mammalian FcER molecule and with
the
specific allergen under conditions suitable for formation of a
FcER:immunoglobulin:allergen complex; and (b) determining the presence of the
immunoglobulin by detecting the complex, the presence of the complex
indicating the
presence of the immunoglobulin.
The present invention also includes a kit for detecting a biologically active,
allergen-specific immunoglobulin in a composition. The kit includes a
mammalian FcER
molecule, the specific allergen, and a means for detecting the immunoglobulin.
In one embodiment, a biologically active, allergen-specific immunoglobulin of
the present invention is heat stable. The present invention also includes an
isolated
biologically active, allergen-specific immunoglobulin that is heat stable and
that
selectively binds to a mammalian FcER molecule.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows a comparison of results obtained from testing a chronic rhinitis
patient with the FcER molecule-based assay, CAP and Immunodot anti-IgE
monoclonal
antibody assays, a histamine release assay and a skin test.
Fig. 2 compares the reactivities of the sera of patients with allergy symptoms
using a FcER molecule-based assay and an anti-IgE monoclonal antibody-based
assay.
Fig. 3 depicts the correlation between allergen specificities detected by heat-
stable FcER a chain-reactive immunoglobulins and those detected by a pan anti-
IgG
monoclonal antibody.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes a method to detect a biologically active
immunoglobulin that selectively binds to a specific allergen in a mammal. The
method
includes the steps of (a) contacting a putative biologically active, allergen-
specific
immunoglobulin-containing composition from a mammal with an isolated FcE
receptor
(FcER) molecule and with the specific allergen under conditions suitable for
formation of
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-4-
a FcER:immunoglobulin:allergen complex; and (b) determining the presence of
the
immunoglobulin by detecting the camplex. The presence of a complex indicates
the
presence of such an immunoglobulin in the composition. As such, included in
the
present invention is the observation that a FcER molecule of the present
invention can
indeed bind to an immunoglobulin bound to its specific allergen. The present
invention
also includes the surprising discovery that such a method permits the
detection of
biologically active, allergen-specific immunoglobulins that are not
detectable, or only
weakly detectable, by a process using anti-IgE antibodies. That is, the
present invention
detects allergen-specific immunoglobulins that in vitro assays using (e.g.,
employing)
antibodies raised against the constant region of IgE are not able to detect
(e.g., identify,
find, observe), or are only able to detect weakly. While not being bound by
theory, it is
believed that such anti-IgE antibodies are not able to recognize or bind to
such
immunoglobulins, at least not to an extent that permits their detection to any
significantly extent (i.e., there may be no detection or only weak detection
of such
immunoglobulins). In studies conducted to date, compositions isolated from
(i.e.,
samples collected from) at least about 5% of a population of mammals include
biologically active, allergen-specific immunoglobulins not detectable by a
process using
anti-IgE antibodies. Preferably, compositions isolated from about 10% of a
population
of mammals include biologically active, allergen-specific immunoglobulins not
detectable by a process using anti-IgE antibodies.
As used herein, a biologically active, allergen-specific immunoglobulin is an
immunoglobulin, or antibody, that selectively binds to a specific allergen and
thereby is
able to activate a FcERI present on a cell surface. As such, the term
biologically active
refers to the immunoglobulin's ability, upon binding to a specific allergen,
to trigger
basophil and/or mast cell degranulation, to activate a cell leading to
mediator release
therefrom, to elicit immediate skin reactivity, and/or to cause other
manifestations of
allergy. A specific allergen is an allergen that selectively binds to an
immunoglobulin
that is capable of activating a FcERI. Such an immunoglobulin is referred to
herein as an
allergen-specific immunoglobulin. As used herein, the term "selectively binds
to"
refers to the ability of a first molecule to bind to a second molecule in a
specific, or
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-S-
selective, manner. That is, the first molecule preferentially binds to the
second molecule
as opposed to binding to an unrelated molecule.
An immunoglobulin of the present invention can be IgE or can be IgE-like. An
IgE-like immunoglobulin is an immunoglobulin that is biologically similar to
IgE in that
it can, in the presence of the relevant specific allergen, bind to and
activate FcERI. IgE-
like immunoglobulins are those that are detected by methods of the present
invention but
not by known processes using anti-IgE antibodies. While not being bound by
theory, it
is believed that such immunoglobulins may be members of a subclass of IgE that
are
allergenically relevant but that are not recognized by anti-IgE antibodies or
may be
ixnmunoglobulins of another isotype that are able to interact with a FcERI in
such a
manner as to elicit an allergenic response.
One embodiment of the present invention is the surprising discovery of a heat
stable biologically active, allergen-specific immunoglobulin; such an
immunoglobulin
reacts (i.e., binds to, positively reacts with) a~FcER molecule of the present
invention but
not with an anti-IgE monoclonal antibody. As used herein, a heat stable
immunoglobulin is an immunoglobulin will bind to a FcER molecule after having
been
exposed to conditions that would inactivate heat labile IgE molecules; note
that it is
known to those skilled in the art the IgE molecules are typically heat labile.
An example
of such conditions is exposure of an immunoglobulin to 56°C for 1 hour.
Without being
bound by theory, it is believed that such heat stable biologically active,
allergen-specific
immunoglobulins represent a novel subclass of immunoglobulins, such as a heat-
stable
IgE immunoglobulin or a FcER-reactive IgG immunoglobulin, perhaps an
anaphylactic
immunoglobulin.
A method of the present invention is advantageous for a number of reasons,
including but not limited to the following: such a method detects
immunoglobulins that
are allergen-specific; such a method detects allergenically relevant
immunoglobulins;
such a method does not detect non-biologically active IgE; such a method does
not
detect irrelevant antibodies of other isotypes; and such a method detects
biologically
active, allergen-specific immunoglobulins in patients having a history of
allergic
symptoms, even in patients whose serum does not include IgE detectable by a
process
using anti-IgE antibodies. Furthermore, a method of the present invention does
not
CA 02328079 2000-10-10
PCT/US99/07530
WO 99!51988 .
-6-
require a composition obtained from a mammal for testing to be diluted prior
to use.
Neither are washing steps required. As such, a composition can either be
diluted or not,
and washing can either be conducted or not. As such, the present invention is
further
distinguished from JP Publication 8-101194, ibid., which discusses the
importance of
sample dilution and washing steps to perform the assay disclosed therein.
One embodiment of the present invention is a method to detect a biologically
active, allergen-specific immunoglobulin using an isolated FcER molecule. It
is to be
noted that the term "a" entity or "an" entity refers to one or more of that
entity; for
example, a protein refers to one or more proteins or at least one protein. As
such, the
terms "a" (or "an"), "one or more" and "at least one" can be used
interchangeably herein.
It is also to be noted that the terms "comprising", "including", and "having"
can be used
interchangeably. Furthermore, a compound "selected from the group consisting
of
refers to one or more of the compounds in the list that follows, including
mixtures (i.e.,
combinations) of two or more of the compounds.
According to the present invention, an isolated, or biologically pure, FcER
molecule, is a molecule that has been removed from its natural milieu; e.g.,
an isolated
FcER molecule of the present invention has been separated from the cell with
which it
might naturally occur (e.g., a basophil or mast cell). As such, "isolated" and
"biologically pure" do not necessarily reflect the extent to which the
molecule has been
purified. An isolated FcER molecule of the present invention can be obtained
from its
natural source (e.g., from a basophil or mast cell), can be produced using
recombinant
DNA technology or can be produced by chemical synthesis.
A FcER molecule (also referred to herein as FcER or FcER protein) of the
present
invention can be a full-length protein, a portion of a full-length protein or
any homolog
of such a protein. As used herein, a protein can be a polypeptide or a
peptide. A FcER
molecule of the present invention can include a complete FcERI (i.e., alpha,
beta and
gamma FcER chains), an alpha chain associated with either a beta or gamma
chain, an
alpha FcER chain (also referred to herein as FcER a chain) or a portion of a
FcER a
chain. As such, a FcER molecule includes at least a portion of a FcER a chain
that binds
to IgE, i.e., that is capable of forming an immunocomplex with an IgE constant
region.
A preferred FcER molecule of the present invention includes a soluble FcER a
chain
CA 02328079 2000-10-10
WO 99/51988
_7_
PCT/US99/07530
(i.e., a FcER a chain without a functional transmembrane or cytoplasmic
domain) or a
portion thereof that is capable of binding to IgE. A FcER molecule of the
present
invention preferably binds to IgE with an~affinity of about KA~ 108, more
preferably with
an affinity of about Kp= '1O9 and even more preferably with an affinity of
about KA~ 10'°.
An isolated FcER molecule of the present invention, including a homolog, can
be
identified in a straight-forward manner by the FcER molecule's ability to form
an
immunocomplex with an IgE. Examples of FcER homologs include FcER proteins in
which amino acids have been deleted (e.g., a truncated version of the protein,
such as a
peptide), inserted, inverted, substituted and/or derivatized (e.g., by
glycosylation,
phosphorylation, acetylation, myristoylation, prenylation, palmitoylation,
amidation
and/or addition of glycerophosphatidyl inositol) such that the homolog
includes at least
one epitope capable of forming an immunocomplex with an IgE.
FcER homologs can be the result of natural allelic variation or natural
mutation.
FcER homologs of the present invention can also be produced using techniques
known in
the art including, but not limited to, direct modifications to the protein or
modifications
to the gene encoding the protein using, for example, classic or recombinant.
DNA
techniques to effect random or targeted mutagenesis.
A preferred FcER molecule of the present invention is a mammalian FcER
molecule. Examples of suitable mammalian FcER molecules include, but are not
limited
to, human, feline, canine, equine, bovine, ovine, porcine, rodent, other
companion
animal (i.e., pet), economic food animal or zoo animal FcER molecules, with
human or
companion animal FcER molecules being more preferred. Particularly preferred
are
human, feline, canine, or equine FcER molecules, with human and canine FcER
molecules being even more preferred. Examples of human, feline, canine, and
rat FcER
a chains are disclosed, for example, in US 4,962,035, ibid.; US 5,639,660,
ibid.; Kochan
et al, ibid.; Shimizu et al., ibid.; Pang et al., ibid.; PCT Publication No.
WO 98/23964,
by Frank et al., published June 4, 1998; PCT Publication No. WO 98/27208, by
Frank et
al., published June 25, 1998; and PCT Publication No. WO 98/45707, by Frank et
al.,
published October 15, 1998. Knowing the nucleic acid and amino acid sequences
of
such genes and proteins, respectively, enables one skilled in the art to
identify other FcER
a chains, including soluble FcER a chains, and IgE-binding portions thereof,
including
CA 02328079 2000-10-10
WO 99/51988
_g_
PCT/US99/07530
those from other mammals. Examples of such techniques are disclosed, for
example, by
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Labs
Press, 1989. In one embodiment, a particularly preferred FcER molecule is a
FcER
molecule that is highly labeled and stable. Methods to produce such a molecule
are
disclosed herein.
An isolated FcER molecule of the present invention can be produced by
culturing
a cell capable of expressing the protein under conditions effective to produce
the protein,
and recovering the protein. A preferred cell to culture is a recombinant cell
that is
capable of expressing the protein, the recombinant cell being produced by
transforming a
host cell with one or more nucleic acid molecules encoding a FcER molecule of
the
present invention (i.e., a nucleic acid molecule of the present invention).
Transformation
of a nucleic acid molecule into a cell can be accomplished by any method by
which a
nucleic acid molecule can be inserted into the cell. Transformation techniques
include,
but are not limited to, transfection, electraporation, microinjection,
lipofection,
adsorption, and protoplast fusion. A recombinant cell may remain unicellular
or may
grow into a tissue, organ or a multicellular organism. Transformed nucleic
acid
molecules of the present invention can remain extrachromosomal or can
integrate into
one or more sites within a chromosome of the transformed (i.e., recombinant)
cell in
such a manner that their ability to be expressed is retained. Suitable and
preferred
nucleic acid molecules with which to transform a cell are those that encode
suitable and
preferred FcER molecules as disclosed herein.
Suitable host cells to transform include any cell that can be transformed with
a
nucleic acid molecule encoding a FcER molecule of the present invention. Host
cells can
be either untransformed cells or cells that are already transformed with at
least one
nucleic acid molecule. Host cells of the present invention either can be
endogenously
(i.e., naturally) capable of producing a FcER molecule protein of the present
invention or
can be capable of producing such proteins after being transformed with at
least one
nucleic acid molecule of the present invention. Host cells of the present
invention can
be any cell capable of producing at least one protein of the present
invention, and
include bacterial, fungal (including yeast), insect, other animal and plant
cells.
CA 02328079 2000-10-10
W O 99/51988
-9-
PCT/US99/07530
Preferably, a recombinant cell is transfected with a recombinant molecule of
the
present invention. A recombinant molecule includes at least one of any nucleic
acid
molecule heretofore described operatively linked to at least one of any
transcription
control sequence capable of effectively regulating expression of the nucleic
acid
molecules) in the cell to be transformed.
A FcER molecule of the present invention can include chimeric molecules
including at least a portion of a FcER molecule that binds to an IgE and a
second
molecule that enables the chimeric molecule to be bound to a substrate in such
a manner
that the FcER portion binds to IgE in essentially the same manner as a FcER
molecule that
is not bound to a substrate. An example of a suitable second molecule includes
at least a
portion of an immunoglobuiin molecule or other ligand that either binds
directly to a
substrate or to its complementary ligand immobilized on a substrate.
A FcER molecule of the present invention can be bound to the surface of a cell
expressing the FcER molecule. A preferred FcER-bearing cell is a recombinant
cell
expressing a nucleic acid molecule encoding a FcER a chain of the present
invention.
The present invention also includes FcER mimetopes and use thereof to detect
biologically active, allergen-specific immunoglobulins. In accordance with the
present
invention, a "mimetope" refers to any compound that is able to mimic the
ability of a
FcER molecule to bind to biologically active, allergen-specific
immunoglobulins. A
mimetope can be a peptide that has been modified to decrease its
susceptibility to
degradation but that still retains-binding activity. Other examples of
mimetopes include,
but are not limited to, carbohydrate-based compounds, Lipid-based compounds,
nucleic
acid-based compounds, natural organic compounds, synthetically derived organic
compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments
thereof.
A mimetope can be obtained by, for example, screening libraries of synthetic
compounds
for compounds capable of binding to biologically active, allergen-specific
immunoglobulins. A mimetope can also be obtained by, for example, rational
drug
design. In a rational drug design procedure, the three-dimensional structure
of a
compound of the present invention can be analyzed by, for example, nuclear
magnetic
resonance (NMR) or x-ray crystallography. The three-dimensional structure can
then be
used to predict structures of potential mimetopes by, for example, computer
modeling.
CA 02328079 2000-10-10
WO 99/51988 PCTNS99/07530
-10-
The predicted mimetope structures can then be produced by, for example,
chemical
synthesis, recombinant DNA technology, or by isolating a mimetope from a
natural
source. Specific examples of FcER mimetopes include anti-idiotypic antibodies,
oligonucleotides produced using Selex technology, peptides identified by
random
screening of peptide libraries and proteins identified by phage display
technology.
A FcER molecule of the present invention can be contained in a formulation,
herein referred to as a FcER formulation. For example, a FcER molecule can be
combined with a buffer in which the FcER molecule is solubilized and/or with a
carrier.
Suitable buffers and carriers are known to those skilled in the art. Examples
of suitable
buffers include any buffer in which a FcER molecule can function to
selectively bind to a
biologically active, allergen-specific immunoglobulin, such as, but not
limited to,
phosphate buffered saline, water, saline, phosphate buffer, bicarbonate
buffer, HEPES
buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered saline),
TES
buffer (Tris-EDTA buffered saline}, Tris buffer and TAE buffer (Tris-acetate-
EDTA).
Examples of carriers include, but are not limited to, polymeric matrices,
toxoids, and
serum albumins, such as bovine serum albumin. Carriers can be mixed with a
FcER
molecule or conjugated (i.e., attached) to a FcER molecule in such a manner as
to not
substantially interfere with the ability of the FcER molecule to selectively
bind to a
biologically active, allergen-specific immunoglobulin.
As disclosed herein, a specific allergen of the present invention is an
allergen
that, when it selectively binds to a biologically active, allergen-specific
immunoglobulin
in vivo, triggers an allergic response. Such an allergen can also bind to such
an
immunoglobulin in vitro. A suitable specific allergen is any substance that
can induce
the production of IgE or IgE-like immunoglobulins. Examples of specific
allergens
include, but are not limited to, bacterial allergens, fungal allergens,
endoparasite
allergens, ectoparasite allergens, food allergens, pollen allergens, other
animal allergens
and other plant allergens. Such specific allergens include, but are not
limited to,
bacterial, yeast, fungal, heartworm, other helminth, flea, fly, mosquito,
mite, midge,
biting gnat, lice, bee, wasp, ant, cockroach, true bug, tick, human dander,
cat, dog, cattle,
poultry, swine, sheep, lamb, dust, tree, weed, shrub, grass, wheat, corn,
soybean, peanut,
walnut, rice, egg, milk, and cheese allergens, as well as other pollen
allergens. Preferred
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-11-
specific allergens include, but are not limited to, cat, dog, grass, fescue,
dock, plantain,
firebush, pigweed, ragweed, thistle, rye, olive, hazel, sage, elm, juniper,
pine, aspen,
cocklebur, sorrel, elder, walnut, cottonwood, ash, birch, cedar, oak,
mulberry, olea,
parietaria, mugwort, ribwort, milk, egg, peanut, celery, tomato, hazelnut,
shrimp, wheat,
soja, dust, ash, smut, heartworm, cockroach, flea, Dermatophagoides,
Alternaria,
Aspergillus, Candida, Cladosporium, Fusarium, Helminthosporium, Mucor,
Penicillium, Pullularia, Rhizopus and Tricophyton allergens, such as, but not
limited to,
Johnson grass, Kentucky blue grass, meadow fescue, orchard grass, perennial
rye grass,
redtop grass, Timothy grass, June grass, Bermuda grass, brome grass, curly
dock,
English plantain, Mexican firebush, lamb's quarters, rough pigweed, short
ragweed,
wormwood sage, American elm, common cocklebur, box elder, black walnut,
Eastern
cottonwood, green ash, river birch, red cedar, Japanese cedar, red oak, red
mulberry,
cockroach, Dirofilaria immitis, Ctenocephalides, Dermatophagoides
pteronyssinus,
Dermataphagoides farinae, Alternaria alternata, Aspergillus fumigatus, Candida
albicans, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporium
sativum,
Mucor recemosus, Penicillium notatum, Pullularia pullulans, Rhizopus nigricans
and
Tricophyton spp. A preferred flea allergen is a flea saliva allergen, such as
those flea
saliva products and proteins disclosed in U.S. Patent No. 5,646,115, by Frank
et al,
issued July 8, 1997.
A specific allergen can be produced from its natural source or can be produced
synthetically; a protein allergen can also be produced recombinantly. A
specific allergen
can be a whole allergen or a portion thereof. The smallest portion is an
epitope that is
capable of eliciting an immune response against the whole allergen from which
the
portion is derived.
One embodiment of the present invention is a method to detect a biologically
active immunoglobulin that selectively binds to a specific allergen in a
mammal. The
method includes the steps of (a) contacting a putative biologically active,
allergen-
specific immunoglobulin-containing composition from the mammal with an
isolated
FcER molecule and with the specific allergen under conditions suitable for
formation of a
FcER:immunoglobulin:allergen complex; and (b) determining the presence of the
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-12-
immunoglobulin by detecting the complex, the presence of the complex
indicating the
presence of the immunoglobulin.
Suitable mammals to test include, but are not limited to, humans, dogs (i.e.,
canids), cats (i.e., felids), horses (i.e., equids), cattle, sheep, swine, and
rodents, as well
as other companion animals, food animals, or zoo animals that are mammals.
Preferred
mammals to test include humans and companion animals, with humans, cats, dogs,
and
horses being particularly preferred, and humans and dogs being even more
preferred. As
such, a method of the present invention detects mammalian immunoglobulins,
preferably
human or companion animal immunoglobulins, more preferably human, feline,
canine,
and equine immunoglobulins, and even more preferably human and canine
immunoglobulins. In one embodiment, such a mammalian immunoglobulin is heat
stable, with human or companion animal heat stable immunoglobulins being
preferred,
with human, feline, canine or equine heat stable immunoglobulins being more
preferred,
and with human heat stable immunoglobulins being even more preferred.
One embodiment of the present invention is an isolated biologically active,
allergen-specific immunoglobulin that is heat stable and that selectively
binds to a
mammalian FcER molecule but that preferably does not bind to an anti-IgE
monoclonal
antibody. As used herein, an isolated, or biologically pure, heat stable
immunoglobulin
of the present invention is an immunoglobulin that has been removed from its
natural
milieu. As such, "isolated" and "biologically pure" do not necessarily reflect
the extent
to which the molecule has been purified. An isolated heat stable
immunoglobulin of the
present invention can be obtained from its natural source, can be produced
using
recombinant DNA technology or can be produced by chemical synthesis. In one
embodiment, a heat stable immunoglobulin of the present invention can be
obtained
using a FcER molecule of the present invention.
As used herein, the term "contacting" refers to combining or mixing, in this
case,
a putative biologically active, allergen-specific immunoglobulin-containing
composition
~th a mammalian FcER molecule and a specific allergen. Formation of a complex
between a FcER molecule, a specific allergen and a biologically active,
allergen-specific
immunoglobulin, i.e., a FcER:immunoglobulin:allergen complex, refers to the
ability of
the specific allergen and FcER molecule to selectively bind to the
biologically active,
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-13-
allergen-specific immunoglobulin in order to form a stable complex that can be
measured (i.e., detected). Binding between a FcER molecule, a specific
allergen and a
biologically active, allergen-specific immunoglobulin is effected under
conditions
suitable to form a complex; such conditions (e.g., appropriate concentrations,
buffers,
temperatures, reaction times) as well as methods to optimize such conditions
are known
to those skilled in the art, and examples are disclosed herein. Examples of
complex
formation conditions are also disclosed in, for example, in Sambrook et al.,
ibid. It is to
be noted that a FcER:immunoglobulin:allergen complex can be formed in a
variety of
orders, for example, (a) by contacting the specific allergen with the
composition and
then contacting with the FcER molecule, (b) by contacting the composition with
the FcER
molecule and then with the specific allergen, or (c) by contacting the three
at the same
time.
As used herein, the term "detecting complex formation" refers to determining
if
any complex is formed, i.e., assaying for the presence (i.e., existence) of a
complex. If
1 S complexes are formed, the amount of complexes formed can, but need not be,
determined. Complex formation, or selective binding, between a FcER molecule,
a
specific allergen, and a biologically active, allergen-specific immunoglobulin
in the
composition can be measured (i.e., detected, determined) using a variety of
methods
standard in the art (see, for example, Sambrook et al. ibid.), examples of
which are
disclosed herein.
A putative biologically active, allergen-specific immunoglobulin-containing
composition of the present invention refers to a biological sample from a
mammal. A
suitable biological sample includes, but is not limited to, a bodily fluid
composition or a
cellular composition. A bodily fluid refers to any fluid that can be collected
(i.e.,
obtained) from a mammal, examples of which include, but are not limited to,
blood,
serum, plasma, urine, tears, aqueous humor, central nervous system fluid
(CNF), saliva,
lymph, nasal secretions, milk and feces. A preferred composition of the
present method
is serum.
A composition of the present method can also include a biologically active,
allergen-specific immunoglobulin-producing cell. Such a cell can have such an
immunoglobulin bound to the surface of the cell and/or can secrete such an
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-14-
immunoglobulin. The immunoglobulin can be bound to the surface of a cell
either
directly to the membrane of a cells or bound to a molecule (e.g., an allergen)
bound to
the surface of the cell.
A complex can be detected in a variety of ways including, but not limited to
use
of one or more of the following assays: an enzyme-linked immunoassay, a
radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a
lateral
flow assay, an agglutination assay, a particulate-based assay (e.g., using
particulates such
as, but not limited to, magnetic particles or plastic polymers, such as latex
or polystyrene
beads), an immunoprecipitation assay, a BioCoreTM assay (e.g., using colloidal
gold), an
immunodot assay (e.g., CMG's Immunodot System, Fribourg, Switzerland), and an
immunoblot assay (e.g., a western blot). Such assays are well known to those
skilled in
the art. Assays can be used to give qualitative or quantitative results
depending on how
they are used. Some assays, such as agglutination, particulate separation, and
immunoprecipitation, can be observed visually (e.g., either by eye or by a
machines,
such as a densitometer or spectrophotometer) without the need for a detectable
marker.
In other assays, conjugation (i.e., attachment) of a detectable marker to the
FcER
molecule or to a reagent that selectively binds to the FcER molecule or to the
immunoglobulin being detected (described in more detail below) aids in
detecting
complex formation. Examples of detectable markers include, but are not limited
to, a
radioactive label, a fluorescent label, a chemiluminescent label, a
chromophoric label or
a ligand. A ligand refers to a molecule that binds selectively to another
molecule.
Preferred detectable markers include, but are not limited to, fluorescein, a
radioisotope, a
phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g.,
horseradish
peroxidase), beta-galactosidase, and biotin-related compounds or avidin-
related
compounds (e.g., streptavidin or ImmunoPure~ NeutrAvidin).
In one embodiment, a FcER:immunoglobulin:allergen complex is detected by
contacting a putative biologically active, allergen-specific immunoglobulin-
containing
composition with a specific allergen and with a FcER molecule that is
conjugated to a
detectable marker. A suitable detectable marker to conjugate to a FcER
molecule
includes, but is not Limited to, a radioactive label, a fluorescent label, a
chemiluminescent label, a chromophoric label, or a ligand. A detectable marker
is
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-15-
conjugated to a FcER molecule or a reagent in such a manner as not to block
the ability
of the FcER or reagent to bind to the biologically active, allergen-specific
immunoglobulin being detected. Preferred detectable markers include, but are
not
limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline
phosphatase), biotin,
S avidin, a peroxidase (e.g., horseradish peroxidase), beta-galactosidase, and
biotin-related
compounds or avidin-related compounds (e.g., streptavidin or ImmunoPure~
NeutrAvidin). Preferably, a carbohydrate group of a FcER molecule, and more
preferably of a FcER a chain, is conjugated to biotin. Also preferable is a
FcER molecule
that is highly labeled with a detectable marker.
In another embodiment, a FcER:immunoglobulin:allergen complex is detected by
contacting a putative biologically active, allergen-specific-containing
composition with a
FcER molecule and with a specific allergen and then contacting the complex
with an
indicator molecule. Suitable indicator molecules of the present invention
include
molecules that can bind to the FcER molecule, to the specific allergen, or to
the
immunoglobulin. As such, an indicator molecule can comprise, for example, a
FcER
molecule, an antigen, an antibody or a lectin, depending upon which portion of
the
complex being detected. Preferred identifying labeled compounds that are
antibodies
include, for example, anti-biologically active, allergen-specific
immunoglobulin
antibodies, anti-FcER antibodies, and anti-allergen antibodies. Preferred
lectins include
those lectins that bind to high mannose-containing groups present on a member
of the
complex. More preferred lectins bind to high mannose-containing groups present
on a
FcER molecule of the present invention produced in insect cells. An indicator
molecule
itself can be attached to a detectable marker of the present invention. For
example, an
antibody can be conjugated to biotin, horseradish peroxidase, alkaline
phosphatase or
fluorescein.
In one embodiment, a FcER:immunoglobulin:allergen complex is detected by
contacting the complex with a reagent that selectively binds to a FcER
molecule of the
present invention. Examples of such a reagent include, but are not limited to,
an
antibody that selectively binds to a FcER molecule (referred to herein as an
anti-FcER
antibody) or a compound that selectively binds to a detectable marker
conjugated to a
FcER molecule. FcER molecules conjugated to biotin are preferably detected
using
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-16-
streptavidin, more preferably using ImmunoPure~ NeutrAvidin (available from
Pierce,
Rockford, IL).
In another embodiment, a FcER:immunoglobulin:allergen complex is detected by
contacting the complex with a reagent, such as an antibody or other ligand,
that
selectively binds to the specific allergen to which the biologically active,
allergen-
specific immunoglobulin is bound. Such a reagent may itself contain a
detectable
marker or can be detected with yet another indicator molecule that binds to
that reagent.
The present invention can further comprise one or more layers and/or types of
secondary molecules or other binding molecules capable of detecting the
presence of an
indicator molecule. For example, an untagged (i.e., not conjugated to a
detectable
marker) secondary antibody that selectively binds to an indicator molecule can
be bound
to a tagged (i.e., conjugated to a detectable marker) tertiary antibody that
selectively
binds to the secondary antibody. Suitable secondary antibodies, tertiary
antibodies and
other secondary or tertiary molecules can be selected by those of skill in the
art.
Preferred tertiary molecules can be selected by a skilled artisan based upon
the
characteristics of the secondary molecule. The same strategy is applied for
subsequent
layers.
In one embodiment, a FcER:immunoglobulin:allergen complex can be formed
and detected in solution.
In another embodiment, a FcER:immunoglobulin:allergen complex can be formed
in which one or more members of the complex are immobilized on (e.g., coated
onto) a
substrate. Immobilization techniques are known to those skilled in the art.
Suitable
substrate materials include, but are not limited to, plastic, glass, gel,
celluloid, fabric,
paper, and particulate materials. Examples of substrate materials include, but
are not
limited to, latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF
(poly-
vinylidene-fluoride), and magnetic resin. Suitable shapes for substrate
material include,
but are not limited to, a well (e.g., microtiter dish well), a plate, a
dipstick, a strip, a
bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a
celluloid-type
matrix, a magnetic particle, and other particulates. A particularly preferred
substrate
comprises an ELISA plate, a dipstick, an immunodot strip, a radioimmunoassay
plate, an
agarose bead, a plastic bead, a latex bead, a sponge, a cotton thread, a
plastic chip, an
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-17-
immunoblot membrane and an immunoblot paper. In one embodiment, a substrate,
such
as a particulate, can include a detectable marker.
A preferred method of the present invention includes a step of (a) binding a
FcER molecule to a substrate to form a FcER molecule-coated substrate prior to
contacting the FcER molecule with a putative immunoglobulin-containing
composition
and a specific allergen; (b) binding a specific allergen to a substrate to
form an allergen-
coated substrate prior to contacting the allergen with a putative
immunoglobulin-
containing composition and a FcER molecule; or (c) binding a putative
immunoglobulin-
containing composition to a substrate to form a putative immunoglobulin-
containing
composition-coated substrate prior to contacting the composition with a FcER
molecule
or specific allergen. In the latter case, the substrate can be a non-coated
substrate, but is
preferably a FcER molecule-coated substrate or an allergen-coated substrate.
In a preferred embodiment, a specific allergen is immobilized on a substrate,
such as a microtiter dish well, a dipstick, an immunodot strip, or a lateral
flow apparatus.
Preferred allergens include those disclosed herein. A biological sample
collected from
an animal is applied to the substrate and incubated under conditions suitable
(i.e.,
sufficient) to allow for immunoglobulin:allergen complex formation bound to
the
substrate (i.e., immunoglobulin in the sample binds to allergen immobilized on
the
substrate). A FcER molecule is added to the substrate and incubated to allow
for
formation of a complex between the FcER molecule and the
immunoglobulin:allergen
complex. Preferably, the FcER molecule is conjugated to a detectable marker. A
developing agent is added, if required, and the substrate is submitted to a
detection
device for analysis. In some protocols, washing steps are added after one or
both
complex formation steps in order to remove excess reagents. If such steps are
used, they
involve conditions known to those skilled in the art such that excess reagents
are
removed but the complex is retained on the substrate. Preferred conditions are
disclosed
herein in the Examples section and generally in Sambrook et al., ibid.
In another embodiment, a FcER molecule is immobilized on a substrate, such as
a
microtiter dish well, a dipstick, an immunodot strip, or a lateral flow
apparatus. A
biological sample collected from an animal is applied to the substrate and
incubated
under conditions suitable to allow for FcER:immunoglobulin formation bound to
the
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-18-
substrate. A specific allergen is added to the substrate and incubated to
allow for
formation of a complex between the allergen and the FcER:immunoglobulin
complex.
Preferred allergens are disclosed herein. In one embodiment, the allergen is
conjugated
to a detectable marker. In another embodiment, an indicator molecule,
preferably
conjugated to a detectable marker, that can selectively bind to the allergen
is added to the
substrate. A developing agent is added, if required, and the substrate is
submitted to a
detection device for analysis. In some protocols, washing steps are added
after one or
both complex formation steps in order to remove excess reagents.
A preferred method to detect biologically active, allergen-specific
immunoglobulins is a lateral flow assay, examples of which are disclosed in
U.S. Patent
No. 5,424,193, issued June 13, 1995, by Pronovost et al.; U.S. Patent No.
5,415,994,
issued May 16, 1995, by Imrich et al; WO 94/29696, published December 22,
1994, by
Miller et al.; and WO 94/01775, published January 20, 1994, by Pawlak et al. A
lateral
flow assay is an example of a single-step assay. In one embodiment, a
biological sample
is placed in a lateral flow apparatus that includes the following components:
(a) a
support structure defining a flow path; (b) a labeling reagent comprising a
bead
conjugated to a specific allergen, the labeling reagent being impregnated
within the
support structure in a labeling zone; and (c) a capture reagent comprising a
FcER
molecule. Preferred specific allergens include those disclosed herein. The
capture
reagent is located downstream of the labeling reagent within a capture zone
fluidly
connected to the labeling zone in such a manner that the labeling reagent can
flow from
the labeling zone into the capture zone. The support structure comprises a
material that
does not impede the flow of the beads from the labeling zone to the capture
zone.
Suitable materials for use as a support structure include ionic (i.e., anionic
or cationic)
material. Examples of such a material include, but are not limited to,
nitrocellulose,
PVDF, or carboxymethylcellulose. The support structure defines a flow path
that is
lateral and is divided into zones, namely a labeling zone and a capture zone.
The
apparatus can further comprise a sample receiving zone located along the flow
path,
more preferably upstream of the labeling reagent. The flow path in the support
structure
is created by contacting a portion of the support structure downstream of the
capture
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-19-
zone, preferably at the end of the flow path, to an absorbent capable of
absorbing excess
liquid from the labeling and capture zones.
In this embodiment, the biological sample is applied to the sample receiving
zone
which includes a portion of the support structure. The labeling zone receives
the sample
S from the sample receiving zone which is directed downstream by the flow
path. The
labeling zone comprises the labeling reagent that binds to biologically
active, allergen-
specific immunoglobulins in the sample. A preferred labeling reagent is an
allergen
conjugated, either directly or through a linker, to a plastic bead substrate,
such as to a
latex bead. The substrate also includes a detectable marker, preferably a
colorimetric
marker. Typically, the labeling reagent is impregnated to the support
structure by drying
or lyophilization. The sample structure also comprises a capture zone
downstream of the
labeling zone. The capture zone receives labeling reagent from the labeling
zone which
is directed downstream by the flow path. The capture zone contains the capture
reagent,
in this case a FcER molecule, as disclosed above, that immobilizes the
immunoglobulin
complexed to the allergen in the capture zone. The capture reagent is
preferably fixed to
the support structure by drying or lyophilizing. The labeling reagent
accumulates in the
capture zone and the accumulation is assessed visually or by an optical
detection device.
In another embodiment, a lateral flow apparatus used to detect biologically
active, allergen-specific immunoglobulins includes: (a) a support structure
defining a
flow path; (b) a labeling reagent comprising a FcER molecule as described
above, the
labeling reagent impregnated within the support structure in a labeling zone;
and (c) a
capture reagent comprising a specific allergen, the capture reagent being
located
downstream of the labeling reagent within a capture zone fluidly connected to
the
labeling zone in such a manner that the labeling reagent can flow from the
labeling zone
into the capture zone. The apparatus preferably also includes a sample
receiving zone
located along the flow path, preferably upstream of the labeling reagent. The
apparatus
preferably also includes an absorbent located at the end of the flow path.
Another preferred method to detect biologically active, allergen-specific
immunoglobulins is an immunodot strip assay, such as is employed in a CMG
Immunodot System. In this assay, one or more specific allergens are spotted
onto a
nitrocellulose strip. Preferred allergens include those disclosed herein. A
biological
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-20-
sample collected from an animal is applied to the strip and incubated under
conditions
suitable (i.e., sufficient) to allow for immunoglobulin:allergen complex
formation bound
to the strip. A FcER molecule is added to the strip and incubated to allow for
formation
of a complex between the FcER molecule and the immunoglobulin:allergen
complex.
Preferably, the FcER molecule is conjugated to a detectable marker. A
developing agent
is added, if required, and the substrate is submitted to a detection device
for analysis.
This assay can be a dual-step or multiple-step assay, as desired.
The present invention also includes kits to detect biologically active,
allergen-
specific immunoglobulins based on the disclosed detection methods. One
embodiment
is a kit to detect a biologically active, allergen-specific immunoglobulin
that includes a
mammalian FcER molecule, a specific allergen, and a means for detecting a
biologically
active, allergen-specific immunoglobulin. Suitable and preferred FcER
molecules are
disclosed herein. Suitable means of detection include compounds disclosed
herein that
bind to the FcER molecule, to the allergen, or to the immunoglobulin. As such,
a kit can
also comprise a detectable marker, an antibody that selectively binds to the
FcER
molecule or to the specific allergen, or other indicator molecules.
A kit can comprise one or more specific allergens. If a kit includes two or
more
allergens, the allergens can be in formulations such that the allergens remain
separate or
they can be combined in one or more groups.
A preferred kit of the present invention is one in which the allergens) or the
FcER molecule is immobilized on a substrate. Such a kit can contain one or
more
substrates which can be joined together, for example, in a fan-like format.
The following examples are provided for the purposes of illustration and are
not
intended to limit the scope of the present invention.
EXAMPLES
Example 1
This example describes detection of biologically active, allergen-specific
immunoglobulins in humans using a FcER molecule of the present invention. This
example also compares results obtained using the FcER molecule with those
obtained
using anti-IgE monoclonal antibodies.
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-21-
Sera collected from 188 allergic patients and 53 control patients (patients
who
scored negative by intradermal skin testing) were tested against a variety of
allergens
using FcER a chain-based and anti-IgE monoclonal antibody-based assays. The
FcER a
chain-based assay used PhFcERa,~z-BIOT, a biotinylated human soluble FcER a
chain,
S produced as described in PCT Publication No. WO 98/23964, by Frank et al.,
ibid. The
immunodot strip anti-IgE-monoclonal antibody-based assay used a mixture of
anti-
human IgE monoclonal antibodies TT6H10, available from Immunotech, Marseille,
France, and 4F4, available from CMG; see also Samoilovich et al, ACI News 4,
21-25,
1992. The other anti-IgE monoclonal antibody-based assay was Pharmacia's CAP
assay (CAP is a trademark of Pharmacia-Upjohn), which was performed according
to
manufacturer's protocols.
CMG immunodot strips (available from CMG, Fribourg, Switzerland) were
produced using standard procedures (e.g., Hong et al, J. Immunol. Methods 95,
195-202,
1986; de Weck et al, Rev. F. Allergol. 33, 13-21, 1993) with at least one of
the following
allergens spotted on per strip: Dermatophagoides pteronyssinus,
Dermatophagoides
farinae, Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum,
Penicillium notatum, Candida albicans, cockroach, cat, dog, 6 grass mix, rye,
olive,
birch, oak, hazel, olea, parietaria, Japanese cedar, mugwort, ribwort, milk,
egg, peanut,
celery, tomato, hazelnut, shrimp, wheat, and sofa allergens.
For the FcER a chain-based assay, a sample of one milliliter (ml) of undiluted
serum was incubated for about 2 hours at room temperature with an immunodot
strip
spotted with one or more allergens, after which the strip was washed three
times for
about S minutes each with TBS (50 mM Tris, 150 mM NaCI pH 8.0) at room
temperature. The strip was then incubated for about 1 hour at room temperature
with an
about 1:8000 dilution of PhFcERa,~-BIOT, produced as described above, in one
ml
TBS. The strip was then washed three times for about 5 minutes each with TBS
at room
temperature. The strip was then developed by methods known to those skilled in
the art,
e.g., by incubating with a streptavidin-labeled horse-radish peroxidase for
about 30
minutes at room temperature, washed with TBS three times for about 5 minutes
each at
room temperature, and incubating for 15 minutes at room temperature with an
enzyme
substrate using a method similar to that described in PCT Publication
CA 02328079 2000-10-10
WO 99/51988 1PCT/US99/07530
-22-
No. WO 98/23964, by Frank et al., ibid, except adapted for strips. The strip
was then
washed with distilled water and analyzed.
For the immunodot strip anti-IgE monoclonal antibody-based assay, a sample of
one ml of undiluted serum was incubated for about 2 hours at room temperature
with an
immunodot strip spotted with one or more allergens, after which the strip was
washed
three times for about 5 minutes each with TBS at roomy temperature. The strip
was then
incubated for about 1 hour at room temperature with 1;2000 dilution of 0.5
mg/ml of the
anti-human IgE monoclonal antibody mixture in one nil TBS. The strip was then
washed three times for about 5 minutes each with TBS at rUOm temperature. The
strip
was then developed for about 1 5 minutes at room temperature with a mixture of
2,4
chloronapthol (available from I~~Ierck) and hydrogen peroxide using a method
as
described in Houg et al, ibid. The strip was then washc;d with distilled water
and
analyzed.
Correlations obtained comparing the FcER a chain-based assay, the two anti-IgE
1 S monoclonal antibody-based ass;tys, and skin tests for given allergens arc:
presented in
Tables 1-4. "n" refers to the number of patients. "r" value is the correlation
analysis
value.
Table 1. Comparison of the FcER a chain-based assay and the immunodot
strip anti-IgE monoclonal antibody-based assay for given allergens
~, 4
~, a~ :: ~ r ~.
o ~>~ ~ ~,~'.M~r,,,.
.. .r?ue ~..~...3~..5~'h
9 olea 0.99
9 ~Ilternaria 0.98
81 D. pteronyssinus0.97
81 D. farinae 0.97
81 cat
59 6 grasses 0'.94
mix
59 rye 0.93
81 dog 0.91
59 ribwort 0.77
59 mugwort 0.:57
59 birch O.ll
CA 02328079 2000-10-10
WO 99/51988 I'~CT/US99/07530
-23-
Table 2. Comparison of the FcEEt a chain-based assay and the CAP anti-IgE
monoclonal antibody-based assay for given allergens.
c ,~-.~ s~~ ~ ~ h r ~ > ~, t ;
3'I'' ,~~~.1,?<' M s~x''~~~fl '~
4, f. :.
73 D. pteronyssinus 0.87
58 6 grasses mix 0.70
Table 3. Comparison of the imrnunodot strip anti-IgE monoclonal antibody-
based assay and the C~1P anti-IgE monoclonal antibody-based assay
for given allergens.
Kf 'do- ~r' frxd, T
' ~ ' ~ ii
~f~E~
Y~
73 D. pteronyssinus0.85
57 6 grasses 0.75
mix
Table 4. Comparison of serological assays to skin test:; for given allergens
,~( ~ 4,.~ (M0C / k ~ t ~ - A f, y.(
0 W' SWvM .0 _Y Y / '
~ / ~h y
t~t~
~~rE~
5 , ~
~ ~ ..
~
~ ,~-TXA..b . E.....,.
..F ,..- < .. ;
FcER a chain 73 D. pteronyssinu.s0.41
57 6 grasses mix 0.19
anti-IgE: Immunod~~t73 D. pteronyssinu.s0.31
57 6 grasses mix 0.07
anti-IgE: CAP 73 D. pteronyssinus0.27
57 6 grasses mix 0.17
J
Analysis of the results presented in Tables 1-3 indicates that wExile there is
strong;
correlation between FcER molecule-based assays and anti-IgE antibody assays
for several
of the allergens, there is extremely poor correlation for other allergens. In
many of the
patients showing poor correlatu~n, such poor correlation was due to the
ability of the
FcER molecule-based assay to c(etect biologically active, allergen-specific
immunoglobulins that the anti-:(gE antibody-based ass;iys did not detect.
Furthermore,
Table 4 suggests that the I~cER molecule-based assay correlates better with
skin testing
than do either of the anti-IgE antibody-based assay analyzed.
Table S summarizes results for the sera of 16 patients for which only the Fc~R
molecule-based assay ( FcER) detected immunoglobuli.ns or for which the FcER
molecule-based assay detected a significantly higher level of reactivity than
did the anti~-
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-24-
IgE antibody-based assays (i.e., immunodot (Dot) or CAP); reactivities are
expressed as
arbitrary optical density (O.D.) units. It is to be noted that in the other
allergic patients,
anti-IgE antibody-based assays routinely gave higher reactivities than did the
FcER
molecule-based assay. Those samples in which the FcER molecule-based assay
detected
significant levels of immunoglobulin but the anti-IgE antibody assays detected
little if
any immunoglobulin are highlighted by bolding of the FcER molecule-based assay
results.
CA 02328079 2000-10-10
WO 99/51988 PCTNS99/07530
-25-
Table 5. Comparison of the FcER a chain-based assay and anti-IgE antibody-
based assays for patients exhibiting a reaction against at least one
allergen for which the FcER a chain-based assay showed increased
reactivity
PatientAllergen/Assay
D. D. cat dog cockroach
pteronyssirrus farinae
FcER Dot CAP Fc~R Dot FcER Dot FcER Dot FcER Dot
1375 16 21 61.2 13 19 2 0 2 0 4 1
1392 42 55 100 36 45 15 23 0 0 6 0
349 7 25 0 5 0 0 0 0 0 9 0
6 rye birch mugwort ribwort
grasses
FcER Dot CAP FcER Dot FcER Dot FcER Dot FceR Dot
1520 76 91 100 67 78 45 0 30 4 45 27
832 25 31 16.3 38 22 0 0 2 0 16 5
840 37 48 26.1 28 11 3 0 3 0 12 0
899 47 59 41.8 48 48 0 0 0 0 12 3
922 42 55 36 44 47 5 0 3 0 12 2
408 4 0 3.2 3 0 4 0 3 0 4 0
396 34 49 20.9 26 29 7 0 7 0 16 0
336 35 50 7.5 23 31 5 0 5 0 5 0
273 45 5 6.3 44 5 27 0 13 0 48 0
242 67 98 101 54 76 11 3 12 4 25 19
egg peanut wheat milk
FcERDot CAP FcER Dot CAP FcER Dot CA FcER Dot
P
157 5 18 3 8 0 0 1 0 0
10393 0 0 13 0 4.2 8 6 0 0
1149 2 0 3 0 6 0 25 0 0
The results indicate that at least several of the patients whose sera
demonstrated a
particularly marked reactivity in the FcER molecule-based assay to a specific
allergen
demonstrated such reactivity against more than one allergen; see, for example,
patients
#1375, #349, #1520, #832, #840, #922, #408, #396, #336, #273, #242, and #1149.
One
patient, #273, exhibited biologically active, allergen-specific
immunoglobulins against
each of the specific allergens tested (i.e., 6 grasses mix, rye, birch,
mugwort, and
ribwort) that were detectable with the FcER molecule-based assay but that were
either
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-26-
not detected or only very weakly detected using the anti-IgE antibody-based
assays.
Further testing using allergens from D. pteronyssinus and plantain indicated
that the
patient's biologically active, D. pteronyssinus allergen-specific
immunoglobulins and
biologically active, plantain allergen-specific immunoglobulins were similarly
detected;
see Fig. 1.
Overall, of 140 patients clearly allergic to indoor or outdoor allergens, 13
(i.e.,
9.3%) showed positive results with the FcER molecule-based assay and either
negative or
weak results with the anti-IgE antibody-based assays. Of 53 patients
investigated for
suspicion of allergy but with a negative skin test, 3 (i.e., 5.6%) showed
tested positive
with the FcER molecule-based assay.
In summary, these results indicate that there is a population of allergic
individuals, apparently about 5-10%, that produce biologically active,
allergen-specific
immunoglobulins that are detected by a FcER molecule-based assay but not by
anti-IgE
antibody-based methods.
1 S Example 2
This example describes detection of biologically active, allergen-specific
immunoglobulins in dogs using a FcER molecule of the present invention. This
example
also compares results obtained using the FcER molecule with those obtained
using anti-
IgE monoclonal antibodies.
Sera collected from clinically atopic dogs, experimentally-sensitized dogs,
and
control dogs (dogs who scored negative by intradermal skin testing) were
tested against
a variety of allergens using a FcER a chain-based assay and an immunodot strip
anti-IgE-
monoclonal antibody-based assay as described in Example 1.
In a first study, immunodot strips were prepared as described in Example 1
with
at least one of the following allergens being spotted on per strip: Bermuda
grass, June
grass, Timothy grass, orchard grass, mugwort, rye, fescue, oak, Japanese
cedar, birch,
hazel, plantain, house dust mites, storage mites, cat dander, dat flea,
Alternaria,
ovalbumin, peanut, and milk proteins. When the sera of 15 experimentally-
sensitized
high IgE responder dogs were analyzed for immunoglobulins for specific
allergens using
the FcER a chain-based assay and the immunodot anti-IgE monoclonal antibody-
based
assay, the calculated correlation value was r--0.96. However, when the sera of
111
CA 02328079 2000-10-10
WO 99/51988 PCTNS99/07530
-27-
clinically atopic dogs were analyzed for immunoglobulins for specific
allergens using
the FcER a chain-based assay and the immunodot anti-IgE monoclonal antibody-
based
assay, the calculated correlation value was only r=0.37. The poor correlation
was often
due to the ability of the FcER a chain-based assay to detect biologically
active, allergen-
specific allergens which the anti-IgE antibody-based assay did not detect.
In a second study, an enzyme-linked immunoabsorbent assay (ELISA) was
conducted in a manner similar to that described in PCT Publication No. WO
98/23964,
by Frank et al., ibid., with at least one of the following allergens being
added per well:
Bermuda grass, June grass, Timothy grass, orchard grass, mugwort, rye, fescue,
oak,
cedar, birch, elder, cottonwood, elm, juniper, pine, walnut, aspen, ashmix,
smut, cockle,
dock, sorrel, pigweed, thistle, ragweed, red cedar, house dust mite, EP and
LQ.
Examples of dogs which scored positive with the FcER a chain-based assay but
negative
or weakly with the anti-IgE antibody-based assay are shown in Table 6.
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-28-
U ~ U ~ U ~ U ~ U ~ U ~ U ~ U ~R U ~ U ~ U
u_ ~ v_ u_ ~ u. u_ ~ ~ u. ~ a u_ ~ v_ ~ u_ ~
~a ~ R
vi
c
°°'~° ~3 ~
..: o
x
c .' :,' ;:v:isy:
o ,.~ - H.
N
Co
d
>-,
N
CI th
Ot N p
u> (O 4_ ~
N_ f1 O
.fl 5 . . ir: ~ . ~ .
O ~ ~ o '90' 2G g :rG ' ~ /5 -~t ~ ,E"
° ~ 00 OC7 00 n 00 x.C' - ~' 00
'
o~~o ~~ ~ ~ c~ ,~ ~ w' E
G ~~ ~ :- v:' oc~
O U '0 0 o q ~ 0 0 0
V J :°Y'w.""~ f
° .. V;vax'H
G a
° ~~ ~ r ~c~v ~f~D c~iP
O N ~ . f~ m t~ N tV ~T f'7 ~ 'f'~ U
d 00 CJQ ~ 00 00 O'I'- .-O
04 w g'
Y.
dd oq - do od No 00 ;
p o0 oq ;, 00 0o cJci o0
C
oy N g .~ ~ ~'
cio oq , od ~o Rio ~o ~$.:i'
3 a.
b
O ~ ~ O ~ N ~ ~ N m ~aG' ~ ~N 11 g
U °R °R °~? . oo ~- wir o0
N 4A pp~ O> Ot tpfJ u7 1~' N N t~J p~p :. .
N ~ ~ O 0 0 r 0 ~ ~ ~ G~D ~ ~ m 1f7
oq oq oq o0 o Nr- cio E :,
U
- ~ ~ ~ ~ ~~ :i:
c o o c~ o c~ d o ~ cv ~- o o E :,
U ~ ~v~~ -
~.. E ~o °A ~~ ~ N~ ~c-'~ 'n~ ~c.: ~ x~:~.
s s x <o x~ ~~ v.
oq ocq oq cio .= No oc ~ ,~~ o
Ig ~ o
W ~ ~a
4r ~ N u7 N m - ~ O~ ~ ~ ~ a~0 ~~ C.th "
~1
° N O ~ O O ; ~ ~; N t0 wt Oo
O Cj O G? O Q t 0 0 0 0 .- ~ O G
t0 ~ f~ a~D m N ~ ' ..~. , O r- h- cD ~ N ~ ~:,. N gib
Q' ~ !- O .- O N O '~ N ~ r V .~- Om1 ~. >L> y
O Cj O Q O O ~ O O O O (V r- O G tF~
U ~ a ~;~; ~ F, ~
3 : ;~ s n .-
to ~
NN ~~~ as aa. ~ ~~~ ~e~ .'.,~~,t2'
wm
c''~a ~"' ~i~ ~~ &'8' finGu _ zz v° a n~. 5;'~' ~~~~~,.~~z
E-
CA 02328079 2000-10-10
WO 99/51988 1'CT/US99/07530
-29-
These results indicate that dogs also produce biologically active" allergen-
specific:
immunoglobulins that are detectable using FcfR-based assays but that are not
detectable
using anti-IgE antibody-based assays.
Example 3
This Example describes additional studies to detect biologically active,
allergen-
specific immunoglobulins in humans using a FcER molecule of the present
invention.
This Example also identifies biologically active, allergen-specific
immunoglobulins in
serum samples from a subset of patients that exhibit reactivity with Fc~F~:
molecules but
not with anti-IgE monoclonal antibodies.
Sera collected from 200 lgE positive patients and 50 IgE negative, but
allergen
symptomatic, patients were tested against a variety of indoor- and outdoc:~r
allergens using
a FcER a chain-based assay and ;gin immunodot strip anti-Iglv, monoclonal
antibody-based
assay as described in Example 1. Tables 7 and 8 exhibit the correlation
between the two
assays for indoor (Table 7) and outdoor (Table 8) allergens. Strong
reactivities (OD >
10) are denoted by ''++"; weak r~activities (OD 2-10) are denoted by "-~-"';
and negative
reactivities (OD < 2) are denoted by "-".
Table 7. Correlation between results of a Fc~R a chain-based assay (FcE:R) and
an immunodot strip anti-IgE monoclonal antibody based assay (MAb)
to detect allergen-specific immunoglobulins raised against indoor
allergens
CA 02328079 2000-10-10
WO 99/51988 PCT/US99107530
-30-
Table 8. Correlation between rcaults from a Fc~R a chain-based assay (FcER)
and an immunodot stn:p anti-IgE monoclonal antibody-based assay
(MAb) to detect allergen-specific immunoglobulins raised against
outdoor allergens
Fc,~Et
-t.-~-
a ~ ,- 1672I 4
.~~:,i~!~ 3 22 31
:.ice ~
y : 7 22 265
y ~
xy< ~b.'.~.
~' ~.
~ ~r
xs.. .,.:.
...:<.,~z.l:
Of 337 reactions conducted using indoor allergens, 7 (i.e., '.'.. l%),
exhibited positive
reactivity using the Fc~R a chai;~-based assay and negarive reactivity using
the anti-IgE
monoclonal antibody-based assay. Of 542 reactions conducted using outdoor
allergens,
29 (i.e., 5.3%), exhibited positive reactivity using the FcER a chain-based
assay and
negative reactivity using the anti-IgE monoclonal antibody-based assay. Of the
200
patients, 17 showed such discrepancies in reactivity (i.e;., F<:~R positive
but MAb
negative) for ribwort allergen and 3 showed such discrc;pancies in reactivity
for
cockroach allergen. Such discrepancies were reproducible upon repeated serum
sample
collection from patients and testing, even after several months.
Example 4
This Example demonstrates that a certain subset of patients with allergy
symptoms displays positive reacaivity in a FcER a chain:-based assay but
displays no
reactivity in anti-IgE monoclonal antibody-based assays.
Serum samples from fifty patients that had symptoms of allergy but that scored
negative when tested using an anti-IgE monoclonal antibody-based assay (i.e.,
RAST)
were tested using a FcER a chain-based assay as described in Example 1.
Allergens used
included outdoor allergens, indoor allergens, two mixtures c7f food
allerl;ens, and mold
(mould) allergens. Samples from 8 of the 50 patients (i.c., 3 6%) exhibited Fc
fR a chain
positive reactivity for at least fen some of the allergens, as shown in Fig.
2; each patient
is indicated by a patient number. The clinical features of these patients is
outlined in
Table 9.
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-31-
Table 9. Clinical features of patients reactive with a F'cER a chain but not
with anti-IgE monoclonal antibodies
~t:;>. ~ ~
'~x ; f. r ~.ge FeER pos' Sloe: tests~A~~ '~'~
~ CIr~,~ . : . .... . ~,,~"~ '
~.
.. ;.-.. .....
.....
S 731 Perennial rhinitis18 O,FA,FB all nel;. all all
(12) neg. neg.
asthma
S 101 Perennial rhinitis:?0 O,FA,FB all net,. <ill all
I (12) neg. neg.
recidiv. urticaria
S 798 Seasonal rhinitis'~ O,I,FA,FB Cirass pos_<~Il all
(5) neg. neg.
atopic dermatitis,
familial atopy
S 1364Ctu-onic sinusitis53 O,FA,FB Grass pos. all all
(27) neg. neg.
anesthetics
intol.
S 637 Perennial rhinitis28 O,FC (7) Some foods all all
pos. neg. neg.
S 647 Perennial rhinitis70 O,FC (7) ~~ome foodsall ail
pos. neg. neg.
S 1362Eczematid 80 O,FB,FC not done all all
(1 1) neg. neg.
S 744 Rhinitis and 41 O,I,FA,FB a,ll neg. all all
polyposis (5 I ) neg. neg.
aspirin intolerance
'?
TS = TOPSCREEN with anti-I;gE antibodies
O = outdoor allergens; I = indoor allergens; FA/FB/FC = food allergens; Number
in
parenthesis = maximal O.D.
pos. = positive; neg. = negative
These patients are particularly interesting because they have biologically
active, allergen-
specific immunoglobulins in their sera that react with :Fc~R molecules hut not
with anti-
IgE monoclonal antibodies.
Example 5
This Example demonstrates additional properties of biologically active,
allergen-
specific immunoglobulins of the present invention that react with FcER.
molecules but
not with anti-IgE monoclonal antibodies.
Biologically active, allergen-specific immuno~;lobulins from the eight
patients
described in Example 4 were tasted for heat stability, ~i.e., whether the Fc
fR-reactive
immunoglobulins in the sera o;E such patients would continue to be active
(i.e., bind FcER
a chain) after exposure to heating at 56°C for 1 hour, a condition that
typically
inactivates IgE binding activity. It is known, for example, by one skilled in
the art that
the IgE receptor binding domain of IgE immunoglobulins is particularly heat
labile.
CA 02328079 2000-10-10
WO 99/51988 PCT/US99/07530
-32-
The serum of each of the eight patients was shown to contain heat stable
biologically
active, allergen-specific immunoglobulins; i.e., the sera continued to show
positive
reactivity in a FcER a chain-based assay after exposure of the sera to
56°C for 1 hour at
levels similar to that shown by unheated sera.
An experiment was then conducted to determine how the allergen specificities
detected by the heat-stable FcER a chain-reactive immunoglobulins correlated
with the
allergen specificities detected by a pan anti-IgG monoclonal antibody (i.e., a
monoclonal
antibody that recognizes all IgG subclasses; denoted in Fig. 3 as anti-IgG
moAb). The
results are depicted in Fig. 3, which indicates there is a positive
correlation, particularly
for pollen, mite and food allergens, but not for mold (denoted in the figure
as mould)
allergens.
While various embodiments of the present invention have been described in
detail, it is apparent that modifications and adaptations of those embodiments
will occur
to those skilled in the art. It is to be expressly understood, however, that
such
modifications and adaptations are within the scope of the present invention,
as set forth
in the following claims.
