Note: Descriptions are shown in the official language in which they were submitted.
W092/18535 PCT/US92/0~
-1- 2081900 ~
ENDOMETRIAL ANTIGEN, COMPOSITION, TEST ~IT
AND ME~HOD FOR ENDONETRIAL ANTIBODY DE~ERNINATION
Field of the Invention
This invention relates to the detection of
endometriosis, and to protein antigenic fragments,
reagents, diagnostic test kits and compositions useful
therein.
~ackaround of the Inventio~
Endometriosis is a disease state in which
tissues resembling the uterine mucous membrane, or
endometrium tlocated in the lining of the uterus),
multiply in other parts of the body, such as in the
abdominal cavity. This disease is a significant
problem in gynecology. The presence of the abnormal -
tissues can cause abdominal bleedin~, adhesions,
dysmenorrhea and particularly infertility.
Currently, a preliminary diagnosis of
endometriosis is generally made based on a patient's
history of infertility, unexplained pelvic pain or
other known symptoms. Confirmation is carried out
using a surgical procedure termed laparoscopy to obtain
a sample of tissue for biopsy. This is a procedure
which is unpleasant as well as having the usual dangers
associated with invasive procedures.
It has been reported that antibodies to
normal endometrial tissues have been found in the serum
of patients with endometriosis ~see publications noted
in EP-A-0 387 027, published September 12, 1990).
Various antigens have been speculated as
immunologically related to the endometrial antibodies
found in the serum specimens.
In EP-A-0 387 027, endometrial antigens
having various molecular weights were described as
isolated from cultures or culture media obtained from
several epithelial carcinoma cel' lines. Monoclonal
antibodies and immunological reD~ents directed to the
~ . .,; . ' . ' , . ' " . " .: ' . -:, ' ' . ', . , ' . ' ' ., . .. ' . , ~ ' . ' ! ' ', .' ' .'.. : ' '
WO92/18535 PCT/U592/0~
2 0 8 190 0 -2-
antigens are also described. The antibodies and
antigens were then used to detect endometrial
antibodies in patient specimens using various
immunological procedures.
It would be desirable to have additional
antigens which could be used in a sensitive and
accurate assay for endometrial antibodies.
Summary of the Invention
The present invention provides a protein
antigen isolated from the cytoplasm of epithelial
ade~ocarcinoma cells, the antigen selected from the
group consisting of:
a. a fragment having a molecular weight of from
about 63 to about 67 kilodaltons,
lS b. a fragment having a molecular weight of from
about 33 to about 37 kilodaltons,
c. a fragment having a molecular weight of from
about 40 to about 44 kilodaltons
d. a fragment having a molecular weight of from
about 31 to about 35 kilodaltons, and
e. a fragment having a molecular weight of from -
about 57 to about 64 kilodaltons.
These antig~ns can be provided individually
or in admixture in a buffered composition useful for
detecting the presence of endometrial antibodies.
This invention also provides an endometrial
antibody capture reagent comprising one or more of the -
protein endometrial antigens described above attached - -` -
to a water insoluble support.
Additionally, one or more of the antigens can -
be detectably labeled to provide water soluble
endometrial antibody detection reagents.
The antigen or any of the reagents described
above can be provided in a diagnostic test kit along
with an anti-human antibody reactive with an
endometrial antibody.
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WO92/18535 PCT/US92/n~
_3_ 2 O~l 9 ~ 0
A method for determining endometriosis
comprises:
A. contacting a specimen suspected of containing
endometrial antibodies with a protein antigen as
described above, and
B. detecting any resulting complex of the
antigen with the endometrial antibodies as an
indication of the endometrial antibodies in the `~
specimen.
The present invention provides an
advantageous means for detecting endometriosis without
the need for invasive laparoscopy. This result is
achieved using novel protein antigen fragments isolated
from the cytoplasm of epithelial adenocarcinoma cells
to detect the presence of endometrial antibodies in a
patient specimen, such as serum. Sensitive detection
of the antibodies can be carried out using various
assay formats, as described below.
~rief Descri~tion of the Fiaure
The FIGURE is a photographic image of several
nitrocellulose strips used in an immunoblot assay, as
described in more detail in Example S below.
Detailed Descr;ot;on of the Tnvention
The antigens of this invention are identified
generally by molecular weight in kilodaltons. They are
identified in a narrow range of molecular weights since
it is standard in the art to have some inherent-
inaccuracy (about 10%) in electrophoretic methods for
molecular weight determination. Some of the antigens
have also been characterized by isoelectric point (pI).
The antigens are generally protein fragments
isolated from larger proteins found in the cytoplasm of
human epithelial adenocarcinoma cells. Included among --
such cells are endometrial, breast and ovary cells.
The antigen fragments identified herein can be isolated
from various cell lines, and may have varying amino
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: . . : . .-: . - . . ~ . . . .. . :
W092/18535 PCT/US92/O~U~
20819~0 _4_
acid compositions even though the molecular weight is
the same. Also contemplated as equivalents of the
naturally occurring antigens isolated from cells are
what are termed ~immunological equivalents~ which are
5 peptides which have the same molecular weight, ~:
isoelectric point and immunological reactivity with the .
antibodies of interest.
Representative isolated fragments of this
invention are listed in Table I below, and can be used
singly or in mixtures in the practice of this
nventlon . .
,TABLE I .
Cell Line Antiqen Fraqment Molecular Weiaht (kD)
RL95-2 A 63-67
~ATCC CRL-1671) ..... .
AN3CA Bl 63-67 ~ -
~ATCC HTB-111) B2 33-37 :.
B3 40-44
.. . .
~4 59-64 : .
HEClA C1 63-67
~ATCC HTB-112) C2 33-37 -
C3 40-44
4 59-64 :
~LE D 31-35 :
(ATCC CRL-1622)
T47D E1 63-67
~ATCC HTB-133) 2 33-37
E3 40-44
E4 59-63 :. - .
E5 57 _ 5 G
W092/18535 PCT/US92/0~ ~
2l~19~0 ::
--5--
CAOV3 F1 63-67
(ATCC HTB-75) F2 33-37
F3 40-44
F4 59-64
It is preferred to use the 33-37 kD, 40-44 kD
and 57-5~ kD fragments noted above, individually or in
S a mixture, with the mixture of two or more fragments
being most preferred.
Endometrial antigens can be isolated by
affinity chromatography of extracts of epithelial ~ -
tissue (such as endometrial tissue) which has been
subjected to extraction reagents such as~detergents.
Antibodies (monoclonal or polyclonal) specific to the
antigens can be used in the chromatography process. : -
The treatment of the tissue extracts by column
purification can be carried out using standard
procedures, for example, those described by Davis et
al, ~an~ Re~. 9~, pp. 6143-6148 ~1986). By -isolated~
is meant that the protein antigen fragment is in an at
least partially purified state compared to its natural
state in the cytoplasm of the epithelial adenocarcinoma
cells, or in any other hum~n fluid or tissue specimen.
Various techniques besides those described herein can
be used to isolate the individual antigen fragments
including electroelution from geis as described, for
example, by Harrington, Method~ of Enzvmoloov 1~, PP
-25 488-495 (1990). -
~ ~ In a preferred process, the antigens can be
-~ obtained from tissue culture cells such as cultures of
epithelial adenocarcinoma cell lines such as those
. .. .
mentioned~in EP-A-0 387 027. Representative useful
cell lines are on deposit with the American Type
Culture Collection t~ockville, Maryland), namely: cell
lines HEClA (ATCC HTB-112), ANnCA ~ATCC HTB-lll), RL95-
~: ;: .' .''
.-
: '.
W092/18535 PCT/US92/O~W
208190~ -6-
2 (ATCC CRL-1671), KLE (ATCC CRL-1622), T47D (ATCC HTB-
133) and CAOV3 (ATCC HTB-75). :
The general procedure for isolating the
antigens from a cell line is as follows: The cells are
grown in the recommended medium to greater than 90%
confluency, followed by homogenization in buffered
saline solution or a solution of tris(hydroxymethyl)
aminomethane, sucrose and protease inhibitor. For
extracts in buffered saline solution, particulate
material is removed by centrifugation and the
superna~ant concentrated. In a preferred embodiment,
for extracts in the sucrose solution, particulate
materials are removed by centrifugation and the
supernatant is spun at lOO,OOO x gravity for 1 hour at
4C, then concentrated. Ex;racts (that is, the
cytoplasmic fraction) are then resolved using SDS-PAGE
electrophoresis for an appropriate time and voltage in
an appropriate buffered system. The resulting proteins
are then transferred to nitrocellulose using standard
immunoblotting techniques described, for example, by
Stott, J. Immun. Methods 11~, pp. 153-187 ~1989). More
details about this procedure are provided in Example 1
below. All the materials used in the extraction
procedure are commercially available.
The isolated antigen or mixture thereof can :-
be supplied in a buffered composition for use in -
various immunological methods. The composition is
generally buffered to a pH of from about 6 to about 8 :-;
using one or more suitable buffers such as phosphate
30 buffered saline solution, tris(hydroxymethyl)amino- -
methane, glycine, 3-(N-morpholino)propanesulfonic acid,
borates, and others known in the art, [for example,
Good et al, Biochem., 5, 467 (1966)], most of which are
commercially av ilable. The amount of antigen in such
a composition can vary widely depending upon its
intended use. The isolated antigen fragments can be
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WO92/18535 PCT/US92/0~
_7_ 20819~0
used in crude form (that is, in admixture with
extraneous cellular materials) or at ~arious levels of
purification.
The antigens described herein can also be
provided as detectably labeled water soluble (or water
suspendible) reagents which have an appropriate label
moiety coupled thereto. Useful labels include those
directly detectable, such as radioisotopes, chromogens,
fluorogens, suspendible magnetic particles, suspendible
dyed polymeric particles, chemiluminescent moieties,
bioluminescent moieties, phosphors and others known in
the art. Labels which are indirectly detectable
through reaction with additional reagents include
enzymes, dye-formers and others known in the art (for
example, in EP-A-0 387 027). Particularly useful
labels include radioisotopes and enzymes. Useful
enzyme labels include peroxidase, alkaline phosphatase,
urease, glucose oxidase, ~S~-galactosidase and others
readily apparent t~ ~ne skilled in the art. Peroxidase
and alkaline phosphatase are preferred.
The label moieties can be coupled to antigen
fragments using standard technology described, for
example, in US-A-4,302,438, Marchalonis, ~issh5m~
11~, pp. 299-305 (1969i Hnatowich et al,
~,I~my~l. Method~ , pp. 147-157 ~1983) and Ssi~ns~,
220, pp. 613-615 ~1983) for radiolabeling, and
Yoshitake et al, Eur.J.Biochem., lQl, 395 (1979), Pesce
et al, Clin.Chem., ~Q, pp. 353-359 ~1974), US-A-
4,302,438, US-A-4,376,110 and US-RE-31,006 and
references mentioned therein, for example, for
labeling with enzymes. Antigens can be coupled to -
magnetic or magnetizable particles using the teaching
of, for example, US-A-4,795,698. Chemiluminescent
moieties can be coupled to antigens according to the
teaching of, for example, US-A-4,380,580. Dyed or
fluorescent particles are useful as labels and can be
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: - : : :: . : - - : --:: . - - .: . , . : : . . ,
W092J1853s PCT/US92~0~
20819~0 -8-
attached to antigen according to US-A-4,259,313, EP-A-0 ~ `
208 556 and EP-A-308 235. Fluoroscein or other
fluorescent moities can be attached as a label using
known procedures.
The antigen can also be labeled with a
specific binding moiety that is not specific for
endometrial antibodies. Such moieties include avidin,
biotin, a lectin, a sugar and others readily apparent
to one skilled in the art. The moiety would be
reactive with its corresponding receptor which can be
labeled with an enzyme radioisotope or other moiety as
described above. For example, if the antigen is
labeled with biotin, the corresponding receptor,
avidin, can be coupled to an enzyme. Such labeling
techniques are described, for example, in US-A-
4,496,654, EP-A-0 201 079 and EP-A-0 370 694. :
For particles used in this reagent to be
water suspendible, normally the~ are less than about 1
mmeters in size so that they stay suspended in water
20 for at least 3 hours with little or no agitation. ,
The antigen of this invention can also be
coupled with water insoluble supports to provide
endometrial antibody capture reagents for complexing
with the endometrial antibody, thereby ^capturing~ the
antibody. Any useful support can be used as long as it
is not readily suspendible in water (unlike the
reagents described above) and does not interfere with
the antibody~antigen reaction or any other reactions
necessary for detection of that immunological reaction.
Useful supports include particles of organic and
inorganic polymers, glass, ceramics, silica gel,
metals, metal oxides, filters of paper, glass, matted
fibers and particulate structures, microporous ~ ~-
polymeric filters, gels, microtiter plates, test tubes, -
test cups, vials and others readily apparent to one
skilled in the art. The particles are generally -
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WO92J1853~ PCT/US92/0~
2081900
greater than about 0.05 m meters in diameter. Useful
materials for such supports would also be apparent to
one skilled in the art particularly in view of the
teaching in EP-A-0 387 027. The antigens can be
attached to such materials by adsorption or other non-
covalent means (see for example US-A-4,528,267) or
covalent means using techniques generally known,
including those described above for coupling
particulate labels to antigen, and others described by
Chibata, Immobilized Enzvmes, Halsted Press: New York
(1978) and Cautrecasas, J.Bio.Chem., 245, 1059 ~1970).
Thus, coupling can be directly to the support through
reactive groups or ionic bonds, or through coupling
moieties or proteins as is known in the art. For
example, the antigens can be coupled to microtiter
plates by non-specific adsorption or covalent coupling
to a reacti~e group on the plate.
Antibodies to the antigens of this in~ention
can be deve~oped using standard technology. For
example, polyclonal antibodies can be prepared using
suitable mammals, such as goats, monkeys, rabbits,
guinea pigs and horses. The resulting antisera can be
purified using conventional affinity chromatography
such as described by Mishell et al, Se1ected Methods in
Cellular Immunoloo~, San Francisco, Freeman (1980).
Non-human monoclonal antibodies can also be
prepared using the standard method of Kohler et al, :
Nature, 256, pp. 495-497 (1975) involving the use of
hybridomas prepared from immunized mice or rats to
produce suspended spleen cells. Suitable hybridomas
are available from either commercial sources or various
cell culture collections including the ATCC.
Other types of antibodies, including human
monoclonal antibodies specific to the antigen and
antiidiotypic antibodies can be prepared using the
procedures described in more detail in EP-A-0 387 027.
W092/l8535 PCT/US92/~
2~819~0 The antigens of this invention are useful for
the detection (that is, measuring the presence, amount
or both) of endometrial antibodies found in human body
fluids, such as whole blood, blood serum, suspensions
of endometrial tissues, peritoneal fluid and uterine
fluid or secretions. Preferably, the antibodies are
detected in blood serum. These antibodies are
generally identified as human IgG type antibodies
although IgA type antibodies may also be present.
Detection of endometrial antibodies can be
carried out using a variety of immunological methods,
all of which are generally well known in the art as ~
involving the preferential binding of the antigens of ;
this invention with the corresponding endometrial -
antibodies. Such methods include, but are not limited
to, competitive binding assays, enzyme-linked
immunosorbent immunoassays (ELISA), radioimmunoassays
(RIA), immunometric assays ~sandwich), immunoblots,
agglutination assays, light scattering assays and
ultrasonic probe assays.
Immunoblots can be carried out using standard
procedures described, for example, by Stott (noted
above). Generally, the antigen is transferred to an
immunoblot medium such as nitrocellulose (which is
preferred), ~ylon or polyvinylidine difluoride,
nonspecific sites are blocked with appropriate
materials, and the patient sample is brought into
contact with the medium for a sufficient period of time -
and temperature for antibodies in the sample to complex
30 ~with antigen in the medium. Following washing, the ~-
complex in the medium can be contacted with detectably -
labeled antigen which can Usandwich~ the antibodies, or
with detectably labeled anti-human antibodies whlch are
reactive with the endometrial antibodies. A
representative immunoblot is described in Example 5
below, and the results shown in the Figure.
WO92/18535 PCT/US92/0~
2081900
--11--
Light scattering assays are useful to detect
endometrial antibodies using the antigens of this
invention according to the teaching of EP-A-0 387 027.
Competitive binding assays generally involve
contacting the specimen suspected of containing
endometrial antibodies with the antigen and a known
quantity of labeled endometrial antibodies. The
labeled and unlabeled antibodies compete to complex
with the antigen, and the amount of detectable signal
from the complex is inversely proportional to the
amount of unlabeled antibodies in the specimen. The
labeled antibodies can be prepared using materials and
procedures described generally above for labeled
antigen. Further details of such assays can be
obtained by consulting the considerable literature in
this art, including US-A-3,654,090.
Another type of immunoassay is what is known
as an immunometric or sandwich assay in which the
targeted endometrial antibodies are ~sandwiched~
between specific binding materials. In one embodiment,
the specific binding materials both comprise
endometrial antigen, one being detectably labeled (that
is a detection reagent), and the other being a capture
reagent as described above. In another embodiment one
specific binding material in the sandwich can be either
a capture or detection reagent comprising an antigen as
described herein, and the other is a capture or labeled
anti-human antibody reactive with the endometrial
antibodies. The anti-h~man antibodies are
advantageously labèled with a fluorogen, enzyme or
radioisotope. Where the anti-human antibody is labeled
with an enzyme, the assay is known as an ELISA.
Specific details about such assays are well known in
the art, including US-RE-32,696, US-A-4,376,110 and US- --
35 -A-4,486,53~. Anti-human antibodies reactive with the
~ ' '
:
:.
WOg2/18535 PCT/US92/0~ ~
2081900
-12-
endometrial antibodies can be prepared using standard
techniques. Many are commercially available.
Endometrial antibodies can be isolated from
serum or other patient specimens by transferring an
antigen fragment of this invention to nitrocellulose,
contacting the immobilized antigen with the patient
sample to complex the endometrial antibodies, and
eluting the antibodies off the nitrocellulose using a -
high salt or low pH solution according to standard
procedures.
In many of the assays described above,
various wash solutions, blocking solutions, and dye-
providing solutions ~if the label is an enzyme or other
chemical requiring further reaction fdr detection) may
be needed. The details of such materials would be
readily ~pparent to one skilled in the art having ;
relevant publications at hand. Obviously, the specific -
reagents used would be dependent upon the form of assay
and labels used therein.
Patient samples, such as serum samples, can
be diluted if desired with water, buffer or suitable
diluents commercially available for that purpose.
Particularly useful diluent compositions are described
in EP-A-0 337 785 ~published October 18, 1989).
~he assays can be carried out in appropriate
equipment or test devices. Immunoblots, for exa~ple,
are carried out using appropriate media, such as
nitrocellulose strips. Competitive binding and
sandwich assays can be carried out using microtiter
plates having a multiplicity of test wells, test tubes,
test slides, disposable test devices such as those ~-
described in US-A-3,825,410, US-A-3,888,629, US-A-
3,970,429, US-A-4,446,232, US-A-4,870,007, US-A-
4,921,677 and US-A-4,948,561, and other containers
readily apparent to one s~illed in the art. Preferred
test devices include micro-titer plates and disposable
- . . . , . .. : . .
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- . - .: : . . . :. : . ` .:
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WO92/18535 P~T/US92/0~
-13- 2~8~900
test devices (commercially available in SURECELLTM test
kits marketed by Eastman Kodak Company) having
microporous membrane disposed therein for separating
complexed materials from uncomplexed materials.
The antigens, compositions or reagents of
this invention can be supplied individually or as part
of a diagnostic test kit. Such kits may include the
compositions and reagents (noted above) used in
particular assays as well as the necessary
instructions, test devices, specimen handling equipment
for assaying one or more specimens. Preferably, the
kit includes the antigen (or mixture thereof), labeled
anti-human antibodies reactive with the endometrial
antibodies, and a means for detecting the resulting `
lS immunological reaction. The detecting means can be a
test device, microtiter plate, a dye-providing
composition or others known in the art, or a
combination thereof.
The following examples are presented here to
illustrate the practice of this invention. They are
not meant to be limiting in the scope or specific
embodiments. Unless otherwise indicated, the
percentages are by weight.
Example 1: _
The following procedure and materials were
used to isolate several antigen fragments using various
epithelial adenocarcinoma cell lines.
Six cell lines: HEClA ~ATCC HTB-112), AN3CA
(ATCC HTB-lll), RL95-2 ~ATCC CRL-1671), KLE (ATCC CRL-
1622), T47D (ATCC HTB 133) and CAOV3 ~ATCC HTB-75) were -
obtained from the American Type Culture Collection
(Rockville, Maryland).
Each cell line was treated in the following
manner: it was grown in recommended media ~for example, -~
commercially available McCoy's med~a for ~EClA) to
gre~ter than 90% confluency ~at this level, there is -
, ., : .:
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WO92/18535 PCT/US92/02888
2~81990 -14-
confidence that one or more of the antigens are
present). The resulting cells were homogenized in a
solution of tris(hydroxymethyl)aminomethane buffer (50
mmolar, pH 7.4), sucrose (250 mmolar) and protease
inhibitor (a mixture of 0.5 ~g/ml of leupeptin, 0.7
~g/~l of pepstatin, 372 ~g/ml of EDTA Na2 and 2 ~g/ml
of aprotinin available from soehringer-Mannheim or
Sigma Chemical) for 2 minutes at 4C using a mechanical -
homogenizer. Cellular debris, including nuclei,
mitochondria and unlysed cells, was removed by
centrifugation, followed by ultracentrifugation at
100,000 x gravity for 1 hour at 4C to remove all
unwanted cellular debris and leaving the contents of
the cytoplasm. The supernatant was then concentrated -
lS using a Centricell concentrator (30,000 normal
molecular weight limit, Polysciences, Warrington, Pa.)
to about 0.5-10 mg/ml protein.
The extracts were resolved using SDS-PAGE
electrophoresis with a 10% uniform polyacrylamide
reducing gel in a buffer solution of
tris(hydroxymethyl)aminomethane buffer (25 mmolar, pH
8.5), glycine buffer (200 mmolar), sodium
dodecylsulfate (0.1~) and sodium acetate ~100 mmolar)
for 3-4 hours, increasing the voltage to 400 volts.
Isoelectric point (pI) was determined by two-
dimensional electrophoresis. The cell lysate proteins,
prepared as described above, were first separated by
isoelectric point using isoelectric focusing in the
first dimension, and then the electrofocused proteins
were separated according to molecular weight by SDS-
PAGE electrophoresis in the second dimension. The
proteins were blotted to nitrocellulose as described in
Stott, J.Immun. Methods, 119, pp. 153-187, (1989). The
blotted protein was probed with patient serum. Sera
35 believed to contain endometrial antibodies (as ~ -
determined by indirect immunofluorescence microscopy)
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WO92tl8535 PCT/US92/O~U~ ~
-1S- 2081900 ~
and as being from patients known to have endometriosis
(as determined by laparoscopy) were tested. Sera known
to be negative for endometrial antibodies using similar --
techniques were also tested as negative controls.
Reactive proteins were developed using a standard
detection system. The positive serum antigens (which
are the 63-67 kD, 40-44 kD, 33-37 kD, 57-59 kD and 59-
64 kD antigens) were compared to the negative serum
antigens ~,which are common to negative and positive
serum: which may include 30-32 kD, 18-22 kD, 47-50 kD
and 28-30 kD antigens). Antigens unique to the
positive serum were then noted and the molecular
weights and isoelectric points calculated. In other
words, the antigens were identified by subtracting the
common bands on the immunoblots from the bands shown on
the immunoblot for the positive serum sample.
The molecular weight of each fragment was
determined by comparing measured distances (cm) of
target antigens to the measured distances (cm) of a -
resolved mixture of standard SDS-PAGE low molecular
weight protein markers. Molecular weights were
determined and expressed in kilodaltons ~kD) using a
polynomial curve fit calculated using CRICRETGRAPHTM
software (available from Cricket Software, Inc.), and
EXCELTM software (available from Microsoft). Both
programs were run on a Macintosh 2 computer.
The isolated antigen fragments and available
data are listed in Table II below. The molecular
weight of each fragment is listed as a narrow range
because the exact value is difficult to determine using
known procedures and a 10% variation is generally -
accepted in the art.
T A B L E II
.. ..
Antigen Cell Line Molecular Isoelectric
Fraament Source Weiqht (kD)** Point @
-- .
'~ .,
... . . . . . ....... .. .. . . 1 . .. . .. .. . . . . . . .. . . .. .
': '', " , . I' , ' ' . , ,' ~: '' ' , ,' .
.. , . - , ,. - . .... , . " ... .... ... . . ..
WO92/18535 PCT/US92/02~ ~
2081900
-16-
A RL95-2 63-67 NA* :
Bl AN3CA 63-67 NA :~
B2 AN3CA 33-37 NA ~ :
3 AN3CA 40-44 NA
4 AN3CA 59-64 NA
Cl HEClA 63-67 4.5
,' - .
~2 HEClA 33-37 5.8
C3 HEClA 40-44 4.6 `
C4 HEClA 59-64 6.0
El T47D 63-67 4.5
E2 T47D 33-37 NA
E3 T47D 40-44 4.6
E4 T47D 59-64 NA
E5 T47D 57-59 NA
Fl CAOV3 63-67 4.5
2 CAOV3 33-37 5.5
3 CAOV3 40-44 4.6
4 CAOV3 59-64 6.0
*NA = not available
** kD = kilodaltons
~ values reflect plus or minus 1.0
Example 2: ~ffered ~om~ositions of Antiaens
one or more of the anti~ens were added to
tris(hydroxymethyl)aminomethane buffer ~pH 8.3) to form
~ a crude buffered composition of this invention. These
compositions can be stored in suitable containers, for -
example in test kits, until their use or immobilization
on solid supports for use in assays.
Example 3: Pre~aration of netectablv Tabeled
Endometrial Antibodv Reaaent
This is a prospective example of how a
detectably labeled endometrial antibody reagent of this
invention can be prepared. This reagent would comprise
an endometrial antigen fragment (isolated as described
. .
::. .
~ .
WO92/18535 P~T/US92/0~
~0~1900
. .
above) which is labeled with 5-dimethylamino-
naphthalene-l-sulfonyl chloride, a fluorescent moiety.
The reagent could be prepared by adding a 3-
to 5-fold molar excess of 5-dimethylaminonaphthalene-l-
sulfonyl chloride to isolated antigen fragment intrls(hydroxymethyl)aminomethane buffer (25 mmolar, pH
8.3). Before the addition, the chloride is dissolved
in acetone equal to l~ of the final volume. Reaction
would proceed for l to 2 hours at room temperature, and
the resulting mixture dialyzed in the buffer (25
mmolar, pH 8.3) to provide the desired reagent. ;
Example 4: Pre~aration of Endometrial Antibodv
Caoture Reaaent
This is an example of the preparation of an
endometrial antibody capture reagent of this invention.
A mixture of endometrial antigens (El, E2 E4
and Es) were isolated as described in Example l. The
antigen fragmen~s were further purified as follows:
Extraneous proteins were removed by
precipitating them at 25% ammonium sulfate and
centrifugation. The pellet was discarded, and the
supernatant containing the antigen fragments was
treated by adding ammonium sulfate to 40% and
centrifugation, and the resulting pellet was
resuspended in tris(hydroxymethyl)aminomethane buffer
~pH 7.5). This suspension was dialyzed against buffer.
The antigens were was further purified by i
anion-exchange chromatography using a Waters PROTEIN-
PAKTM DEAE column. The antigen fragments were then
eluted from the column using tris~hydroxymethyl)amino-
methane buffer (0.02 molar, pH 8) and a sodium chloride
gradient. The antigens eluted at between 0.3 and 0.45
molar sodium chloride.
The resulting solution was diluted to 30 ~g
protein/ml in phosphate buffered saline solution
containing protease inhibitors, and a sample (l00
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WO92/18~35 PCT/US92/02
-18-
~ i~ 0~ was added to each well of a polystyrene microtiter
2 plate and incubated at room temperature for two hours.
The plate was then washed three times with phosphate
buffered saline solution. Remaining binding sites on
the plates were blocked with bovine serum albumin (3%)
in phosphate buffered saline solution for two hours at
room temperature. The plate was then washed three
times with a solution of TWEENTM 20 nonionic surfactant
(0.05%) in phosphate buffered saline solution.
Serum samples were diluted at 1:5 or 1:10 in
a diluent of TWEENTM 20 (0.05~) and bovine serum
albumin (3%) in phosphate buffered saline solution.
The diluted samples were incubated in the plate wells
for two hours at room temperature while being shaken.
The wells were washed three time with the wash solution
containing TWEENTM 20.
Goat anti-human immunoglobumin F(ab')2
fragments conjugated to horseradish peroxidase, diluted
in the diluent and filtered through a 0.2 micrometer ~ -
filter, were added to the wells and allowed to react
for 1 hour at room temperature.
The plate was again washed three times, and a
dye-providing composition (200 ~1) was added to each
well and allowed to react for about five minutes, This
25 compo9ition included 2-~4-hydroxy-3-methoxyphenyl)-4,5- ;
bis(4-methoxyphenyl)imidazole leuco dye ~0.2 mmolar)
poly(vinylpyrrolidone) (1.25 %), 4'-hydroxyacetanilide
(5 mmolar), diethylenetriaminepentaacetic acid chelator
(0.01 mmolar) and hydrogen peroxide (8 mmolar) in
sodium phosphate buffer (10 mmolar, pH 6.8).
A solution (100 ~1) to stop dye formation was
added and the dye density was evaluated. Test samples
were evaluated using a calibration curve generated from
known positive and negative serum samples.
Example 5: Detection of Endometrial Antibodiesin
Patient Specimens
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WO92/18535 PCT/US92/~W~
20~1900
This example demonstrates the use of the
isolated endometrial antigens to detect endometrial
antibodies present in patient blood sera by
immunoblotting techniques.
The antigen fragments described in Example 1
were transferred to nitrocellulose strips using -
optimized immunoblotting techniques as described by
Stott, su~ra. The buffer for transfer was composed of
tris~hydroxymethyl)aminomethane buffer (25 mmolar, pH
8.3), glycine (200 mmolar) and methanol (20%, HPLC
grade). To carry out the transfer, 75 volts at 4C
were applied for 2 hours. Upon completion of the
transfer, nonspecific protein binding sites were
blocked for one hour at 24C with a "blockingR solution
of tris(hydroxymethyl)aminomethane (20 mmolar, pH 7.5)
containing gelatin (3~), normal goat serum (0.4%) and
sodium chloride (500 mmolar). The blocked
nitrocellulose was then washed twice, 5 minutes each
time, with tris~hydroxymethyl)aminomethane (20 mmolar, ;~
pH 7.5) containing sodium chloride (500 mmolar) and
TWEENTM 20 nonionic surfactant (0.05%). The
nitrocellulose strips and diluted serum samples (10 ml)
were then contacted for incubation for two hours at
24C. The serum had been diluted 100-fold in
tris(hydroxymethyl)aminomethane (20 mmolar, pH 7.5)
containing gelatin (1%), sodium chloride (500 mmolar), ~
and TWEENTM 20 nonionic surfactant (0.05%). -
Sera bel-ieved to contain endometrial -
antibodies (as determined by indirect
30 immunofluorescence microscopy) and as being from : -
patients ~nown to have endometriosis (as,determined by
laparoscopy) were tested. Sera known to be negative
for endometrial antibodies using similar techniques ~
were also tested as negative Controls. Sera was also - -
35 tested without prior knowledge of the presence or -
absen~e of endometrisis (~blind~ studies). The results
WO92/1853~ PCT/US92/0
208 19 ~0 of these "blind~l studies are noted in Table IV below by
an asterisk (*).
After serum incubation, the nitrocellulose
strips were washed four times (5 minutes each time)
S with the buffered solution containing TWEENTM 20 noted
above (30 ml) to remove uncomplexed materials.
The strips were then incubated for two hours
at 24C in contact with anti-human antibodies (13.2
of conjug~te in 40 ml of solution used to dilute
patient serum) directed to the serum endometrial
antibodies bound to the immobilized transferred antigen
fragments. The anti-human antibodies were comprised of
goat anti-human IgG (heavy and light chain) antibodies
labeled with alkaline phosphatase for detection. The
conjugate was purchased as part of an IMMUN-BLOTTM
assay kit (BioRad Laboratories). Upon completion of
the antibody-conjugate incubation, the nitrocellulose ;
strips were washed four times (S minutes each) with the
buffered TWEEN~M 20 solution noted above, and once (S
minutes) with the buffer solution noted above without
TWEENTM 20 to remove uncomplexed reactants and excess
TWEENTM 20 nonionic surfactant.
To detect the resulting bands in the strips,
a dilute solution of 5-bromo-4-chloro-3-indolyl
phosphate and nitroblue tetrazolium lS00 ~1 of each
reagent stoc~ in 50 ml of 0.1 molar
tris(hydroxymethyl)aminomethane buffer, pH 9.5] were
added.
-Vpon completion of sufficient color
development of the-bands (usually about 20 minutes),
the substrate was removed and the strips were washed
with deionized, distilled water for 10 minutes to
quench further color formation. Tables III and IV
below show the results of the sera screen using the
35 assay noted above. The antigens used for obtaining the -~
data in Table III were extracted using phosphate
";, ' ' ,. '' . ' : ' ' : ' " ' ' '
WO92/18535 PCT/US92/0
-21- 2~ 0
buffered saline solution while Table IV data were
obtained using antigens extracted using the buffered
sucrose solution described in Example 1 above.
The FIGURE shows the immunoblot bands in ~. .
strips la, lb, 2a and 2b for the C3 (~0-44 kilodalton)
and C2 (33-37 kilodalton) fragments (identified in the
FIGURE as ~42 kD" and ~35 kDn, respectively). Bands
lc, ld, 2c and 2d are negative controls and do not show ;
bands for the noted fragments.
T A B L E III
, : ;
Normal Patients Endometriosis Patients
Antigen (positives/ (positives/
Fra~ment(s)total samoles) total sam~les)
A 0/2 3/3 ~ .
B1 0/2 3t3
2 0/2 , 3/3
C1 1~8 9/11
C2 0/8 9/11
D2 0~2 a~3
.
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WO92/18535 PCT/US92/0
-~
-22-
20~19~ ~
T A s L E IV
Normal Patients Endometriosis Patients
Antigen (positives/ (positives/
Fragment(s)total sam~les)total sam~les)
r *
-2 2/8 20/26
c3* 2/8 19/26
C ~ .
4 1/10 14/16
E2 0/5 5/5
E3 0/5 5/5
F2 0/5 5/5
F3 0/5 5/5
B2 0/4 6/6
B3 0/4 6/6
4 0/7 13/16
* ~Blind" studies . : .
Example 6~ q~ei9~_sf_r~s~8~ nL~bodies Usina
Disposable Test Device
This example demonstrates the detection of
endometrial antibodies using a disposable test device
and the ELISA immunological technique.
Materials:
Non-purified antigen (containing fragments
C1, C2 and C3, identified in Table I above) isolated
from HE~lA cell line las described above) was
covalently attached to particles composed of
poly~styrene-co-3-(~-vinylbenzyl-thio)propionic acid]
(97.59:2.41 molar ratio, 1.4 mm average diameter)
(2.53% solids) to form a particulate endometrial
antibody capture reagent of this invention.
: 20 A particulate negative control reagent was
prepared by similarly immobilizing a-casein on the
same type of particles (2.05% solids).
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W092/18s35 PCT/US92/0~
-23- 2081~00
The disposable test device used was a
SURECELL M test device (Eastman Kodak Company) having a
LOPRODYNE polyamide microporous (5 ~m) membrane (Pall
Corporation) mounted therein. The test device has
three test wells, one for the negative control, and two
for the specimen. The particulate reagents described
above (0.3% final solids) were coated on designated
test well membranes in the test device, and dried
overnight at room temperature.
The serum diluent composition used was
composed of tris(hydroxymethyl)aminomethane (100
mmolar, pH 8.0), succinylated casein (1%), TWEENTM 20
(0.05~) and gum arabic (1%) (available from Cetus
Corporation). This diluent is similar to that
described in EP-A-0 337 785 (noted above).
The wash solution was comprised of 1-methyl-
2-pyrrolidinone (10%), NONIDETTM P-40 (0.1%), sodium
chloride (500 mmolar), TWEENTM 20 (0.25%), sodium
phosphate, monobasic (50 mmolar, pH 7.4) (available
from Cetus Corporation).
The anti-human antibodies used were goat
anti-human IgG ~heavy and light chain) antibodies and
were conjugated with horseradish peroxidase ~available
from Jackson Immunoresearch).
~he leuco dye composition comprised 2-(4- .,.',7':', .
hydroxy-3-methoxyphenyl)-4,5-bis~4-
methoxyphenyl)imidazole leuco dye (0.23 mmolar)
poly~vinylpyrrolidone) ~1.25 ~), 4'-hydroxyacetanilide
~5 mmolar), diethylene-triaminepentaacetic acid
chelator (0.01 mmolar) and hydrogen peroxide (8 mmolar)
in sodium phosphate buffer (10 mmolar, pH 6.8).
The dye stop solution comprised sodium azide
O . 1% ) .
Assav Procedure: ~
.: -
WO92/18535 PCT/US92/02
208 19~ A patient serum sample (25 ~1~ was diluted by
adding it to the serum ~iluent (2 ml) in standard assay
squeeze tubes. Filter tips were attached to the tubes,
and the diluted sample was added to each well of the
test device to the top of the fill dot. Fluid was
allowed to flow through the membranes.
Diluted (1:10,000)-labeled anti-human
antibodies (40 ~1) were added to each test well. Fluid
was allowed to drain through the membranes and the test
was incubated for one minute. Each test well was then
washed twice (250 ~1) with the wash solution.
The dye-providing composition (40 ~1) was
then added to each test well. The fluid was allowed to
drain and the test wàs incubated for 2 minutes. The
dye stop solution ~(80 ~1) was then added, followed by
fluid drainage.
The visual dye signal was evaluated and
scored ~0 being lowest dye signal and 10 being the
highest). Transmission densities were also measured
20 using standard densitometric procedures. The results -
are shown in Table V below.
Wo 92/18535 Pcr/US92/02888
-25- 2~819~0
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W092/1853S PCT/US92/0~8
2~ 900 -26-
The invention has been described in detail
with particular reference to preferred embodiments .
thereof, but it will be understood that variations and
modifications can be effected within the spirit and
scope of the invention. Moreover, all patents, patentapplications (published or unpublished, foreign or
domestic), literature references and other prior art
referred to hereinabove are incorporated herein by
reference for any teaching pertinent to the present
invention.
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