U.S. patent application number 09/260305 was filed with the patent office on 2001-07-05 for use of ligands specific to major histocompatibility complex- class i antigens for diagnosing endometriosis.
This patent application is currently assigned to PROCREA BIOSCIENCES INC.. Invention is credited to LACHAPELLE, MARIE-HELENE, Miron, Pierre, ROY, DENIS-CLAUDE.
Application Number | 20010006782 09/260305 |
Document ID | / |
Family ID | 27002779 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006782 |
Kind Code |
A1 |
Miron, Pierre ; et
al. |
July 5, 2001 |
USE OF LIGANDS SPECIFIC TO MAJOR HISTOCOMPATIBILITY COMPLEX- CLASS
I ANTIGENS FOR DIAGNOSING ENDOMETRIOSIS
Abstract
It is an object of the present invention to provide the
clinicians with a new application for ligands specific to MHC-class
I antigens, especially HLA-ABC antigens, this new application
residing in the detection and diagnosis of endometriosis. It is
also an object of the present invention to provide a method and a
test kit for diagnosing endometriosis, preferably by
immunohistochemistry, using a monoclonal anti-HLA-ABC antibody as a
preferred ligand or diagnostic reagent. This new method is
non-invasive and is more reliable as a screening test than the
conventionally used laparoscopy. When the endometrium of a woman
tests negatively with the claimed method, it prevents the use of
laparoscopy which is an invasive method for detecting
endometriosis. This method can be practised on a specimen obtained
from the endometrium of a patient and does not require a specimen
sampled directly from the endometriotic foci.
Inventors: |
Miron, Pierre; (LAVAL,
CA) ; LACHAPELLE, MARIE-HELENE; (LAVAL, CA) ;
ROY, DENIS-CLAUDE; (LAVAL, CA) |
Correspondence
Address: |
MERCHANT & GOULD
P O BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
PROCREA BIOSCIENCES INC.
QUEBEC
CA
|
Family ID: |
27002779 |
Appl. No.: |
09/260305 |
Filed: |
March 2, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
08860064 |
Sep 18, 1997 |
|
|
|
Current U.S.
Class: |
435/6.17 ;
435/7.21; 435/7.5; 435/7.9; 435/806; 435/960; 435/975 |
Current CPC
Class: |
G01N 33/689 20130101;
G01N 33/56977 20130101; G01N 2474/20 20210801; G01N 2800/364
20130101; G01N 33/6893 20130101; Y10S 435/975 20130101 |
Class at
Publication: |
435/6 ; 435/7.21;
435/7.5; 435/7.9; 435/806; 435/960; 435/975 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 1995 |
US |
PCT/CA95/00730 |
Claims
What is claimed is:
1. A method for diagnosing endometriosis comprising: contacting a
biological sample containing glandular endometrial cells with a
ligand which specifically binds a Major Histocompatibility Complex
(MHC)-class I antigen, a proteic precursor or a protein fragment
thereof, or a messenger RNA or a cDNA to a messenger RNA encoding
said antigen, precursor or fragment; wherein the ligand forms a
complex with said antigen, precursor or fragment, and the presence
of the complex is an indication of the presence of
endometriosis.
2. A method for diagnosing endometriosis which comprises: reacting
a first ligand which specifically binds a Major Histocompatibility
Complex (MHC)-class I antigen, a proteic precursor or a peptide
fragment thereof, with a biological sample containing glandular
endometrial cells to form a complex, said complex being detected by
labelling means, the label being present on said first ligand or
the label being present on a second ligand which binds the first
ligand or the label being present on a first reactive member of
said labelling means, said first member reacting with a second
reactive member present on said first or second ligand; and
detecting the complex, wherein the presence of a percentage of
labelled glandular endometrial cells greater than about 65% is an
indication of the presence of endometriosis.
3. A method according to claim 1 or 2 wherein said MHC-class I
antigen is a Human Leucocyte Antigen (HLA)-ABC surface antigen and
said ligand is an anti-HLA-ABC antibody.
4. A method according to claim 3 wherein said anti-HLA-ABC antibody
is produced by hybridoma PHM4.
5. A method according to any one of claims 1 to 4 wherein said
glandular endometrial cells are isolated from said biological
sample.
6. A method according to any one of claims 1 to 4 wherein said
glandular endometrial cells are identified and numbered by
counter-staining.
7. A method according to any one of claims 1 to 4 wherein said
glandular endometrial cells are identified and numbered by means of
their binding to an anti-cytokeratin antibody.
8. A method according to claim 2 wherein said labelling means is
selected from the group consisting of radioisotopes, fluorochromes,
enzymes, biotin/avidin and electron dense molecules.
9. A test kit for diagnosing endometriosis, comprising: a first
ligand which specifically binds a Major Histocompatibility Complex
(MHC)-class I antigen, a second ligand to cytokeratin, and
labelling means for both first and second ligands.
10. A test kit according to claim 9 wherein said MHC-class I
antigen is a Human Leucocyte Antigen (HLA)-ABC surface antigen and
said first ligand is an anti-HLA-ABC antibody.
11. A test kit according to claim 10 wherein said anti-HLA-ABC
antibody is produced by the hybridoma PHM4.
12. A test kit according to any one of claims 9 to 11, wherein said
second ligand is an anti-cytokeratin antibody.
Description
BACKGROUND OF THE INVENTION
[0001] a) Field of the Invention
[0002] This invention relates to the use of ligands specific to a
Major Histocompatibility Complex (MHC)-class I antigen, especially
an HLA-ABC surface antigen, which is normally exposed at the
surface of cell membranes, and which is therefore present in or on
endometrial cell, for the diagnosis of endometriosis. The detection
of this antigen is carried out according to a process comprising
the reaction of a ligand, preferably an antibody, which is normally
used to detect the presence of a MHC-class I antigen at the surface
of all cells expressing it.
[0003] This invention also relates to a method for the diagnosis of
endometriosis using the same ligand(s).
[0004] b) Description of Prior Art
[0005] Endometriosis is one of the most common disorders
encountered in the field of gynaecology, affecting the health of an
estimated 10 to 15% of women during their reproductive years.
Although not life threatening, endometriosis is often associated
with severe pelvic pain and infertility.
[0006] Endometriosis is classically defined as the presence of
endometrial tissue (i.e. glands and stroma) outside the uterine
cavity which is its normal location. Although various hypothesis
have been proposed for its pathogenesis (i.e. transplantation,
lymphatic and vascular metastasis, or coelomic metaplasia), there
are several lines of evidence to support the idea that retrograde
menstruation and implantation are the primary mode of developing
pelvic endometriosis.
[0007] The implantation theory proposes that viable endometrial
tissue is refluxed through the fallopian tubes and implants on the
peritoneal surface or pelvic organs. Some additional etiologic
factors must be present for the development of endometriosis and
could be implicated in an altered immune function.
[0008] To date, there are no sensitive reliable, non-invasive
methods for the diagnosis of endometriosis.
[0009] Over the last decade, an increasing number of reports
suggest that endometriosis is associated with abnormal immune
function. It was first suggested, in 1980, that changes in humoral
immunity cause endometriosis. The C3 components of the complement
and IgG antibodies have been found in the endometrium of women with
endometriosis, with a reduction in the levels of total complement.
In addition, IgG and IgA antibodies have been already identified in
the ovarian and endometrial tissue of women with endometriosis.
[0010] Recently, a high incidence of autoantibodies to
phospholipids (particularly phosphatidylserine) and to histones and
nucleotides has also been reported (Gleicher, 1987), suggesting
polyclonal B-cell activation in endometriosis. The presence of more
generalized autoantibodies have suggested that endometriosis could
be an autoimmune disease. The evidence for autoimmune involvement
is, however, far from unequivocal and many important questions
remain.
[0011] The possibility that cell-mediated immunity is altered in
women with endometriosis was first suggested in 1980. The
concentration and total number of peritoneal macrophages are
increased in endometriosis as well as their activator status. The
contribution of reactive oxygen metabolites (superoxide anion
(O.sub.2--), hydrogen peroxide (H.sub.2O.sub.2) and singlet oxygen
(.sup.1O.sub.2)) to the damage of adjacent normal tissues in the
presence of endometriosis has been suggested by an increased
chemiluminescence of peritoneal macrophages.
[0012] More recently, a defect in natural killer cell (NK) activity
has been reported. This reduced activity of the NK cells could also
be present locally, at the level of the peritoneal fluid. Moreover,
women with endometriosis show significantly more T-cell
suppressor/cytotoxic in their peritoneal fluid.
[0013] Even though a defect in the activity of the NK cells has
been noted in the presence of endometriosis, an increased
resistance of the endometrial cells to the NK mediated cytotoxicity
was also suggested. The mechanisms involved in this endometrial
resistance have remained unexplained.
[0014] In the last five years, a considerable amount of research
has been directed towards the development of a better diagnostic
method for endometriosis. However, a problem lies in the fact that
women suffering from endometriosis may not exhibit any symptoms
other than infertility or they may only exhibit symptoms that mimic
those of many other gynaecological diseases.
[0015] Hitherto, it has been common to use an invasive surgical
method requiring general anesthesia to diagnose endometriosis, such
as laparoscopy or laparotomy which allows the direct visualization
of the pelvic content. However, there are certain disadvantages in
using this difficult and inaccurate method for diagnosing
endometriosis.
[0016] One such disadvantage of this method is that it is sometimes
difficult to detect cases of minimal endometriosis. In addition,
other disadvantages exist resulting from, for example, the presence
of adhesions which can obscure the direct visualization of the
pelvis, making it impossible to even carry out a laparoscopy; or
from the presence of ovarian endometriomas which are often similar
in appearance to functional ovarian cysts and could result in these
endometriomas being misdiagnosed. Moreover, up to sixteen
descriptive types of endometriosis have been identified recently,
which can make the diagnosis of endometriosis even more difficult.
Furthermore, what is even more worrisome is the fact that certain
microscopic foci of endometriosis, which are not identifiable by
laparoscopy, have been identified and documented using a peritoneal
biopsy by scanning with an electron microscope.
[0017] Therefore, even with an increased use of laparoscopy,
endometriosis remains a frequently underdiagnosed condition.
[0018] In general, imaging techniques such as ultrasonography, CT
scanning, and magnetic resonance imaging (MRI) have limited value
in the diagnosis of endometriosis. They can provide information
about the invasiveness of the disease, but none of these imaging
techniques are able to identify superficial diseases of peritoneal
surfaces. Moreover, the high cost of these procedures rarely
justifies their use for the diagnosis of endometriosis.
[0019] The technique of using antibodies like, for instance,
anti-endometrial antibodies as mentioned hereinabove in a method
for the diagnosis of various diseases is a widespread and well
known technique.
[0020] In U.S. Pat. No. 4,444,744, an improved method is provided
for using radiolabelled antibodies to detect the presence of cell
surface antigens, including the type HLA-A, HLA-B AND HLA-DR
surface antigens located on cancer cells, in order to locate and
diagnose these cancer cells. These highly specific radiolabelled
antibodies against the cell surface antigens are also used in a
method for tumour therapy.
[0021] In U.S. Pat. No. 4,666,845, labelled mouse monoclonal
antibodies, namely MF116, MH94, MD144, MH55, MF61, ME46, and ME195
were used to detect the presence of a plurality of surface antigen
on human endometrial, cervical or uterine cancer cells and to
diagnose these cancer cells.
[0022] These labelled monoclonal antibodies are also used in the
treatment of endometrial, cervical or uterine cancers.
[0023] In Canadian patent publication number 2,081,900, a method of
diagnosing endometriosis is described. This method detects the
presence of particular antibodies which are present in specimens
obtained from patients suffering of endometriosis. The antigens
used as an immunological reagent were isolated from the cytoplasm
of epithelial adenocarcinoma cells. These antigens have been
characterized only by their molecular weight and by their ability
to bind the antibodies to be detected.
[0024] In Canadian patent publication number 2,011,704, two methods
of diagnosing endometriosis are described. One makes use of an
antibody, particularly the MS2B6 monoclonal antibody, to detect
endometrial antigens, while the other makes use of antigens also
isolated from epithelial carcinoma cells to detect the presence of
anti-endometrial antibodies. No attempt is made in this application
to clarify the similarity or the complete identity of the antigens
of both methods neither to clarify the similarity or equivalence of
the MS2B6 antibody with regard to the endometrial antibodies.
Furthermore, the antigens isolated from carcinoma cells are also
grossly characterized by their molecular weight and their ability
to bind the antibodies to be detected.
[0025] The first two patents are directed to the use of monoclonal
antibodies to detect surface antigens present on various cancer
cells, including endometrial cancer cells, and to the diagnosis of
these cancer cells. In all cases, the expression of surface
antigens of cancer cells cannot be considered as being similar to
surface antigens found on endometrial cells of a woman suffering
from endometriosis.
[0026] Therefore, the particular techniques illustrated in these
patent documents could not be used as a method for diagnosing
endometriosis or for detecting the different expression of surface
antigens on endometrial cells of a woman with endometriosis.
[0027] Semino et al (Fertility and Sterility (1995) 64(5): 909-916)
described that major histocompatibility complex class I molecules
may have a role in the genetic control of endometriosis. The tested
endometrial cells were taken from healthy women (control) or form
endometriotic foci (by laparoscopy). These authors suggest that
endometrial cells expressing a high number of MHC class I molecules
on their surface give a protective signal to autologous lymphoid
effector cells. On a very limited panel of five patients, they
observed that a variable number of HLA class I molecules was
expressed by endometriotic cells (ranging from a reduction of 15%
to an enhancement of 30%, when compared to endometrial cells
collected contemporaneously in the uterine mucosa).
[0028] Even though this reference may suggest a role for MHC class
I molecules in endometriosis, it does not teach any correlation
between endometriosis and the number of cells expressing MHC class
I molecules. It does not further teach a non-invasive method e.g. a
diagnostic method wherein laparoscopy would be avoided, in other
words, performed on any endometrial cell sample.
[0029] In light of these results, there is clearly a great need for
a non-invasive diagnostic test which is more reliable than the
previous techniques and which is easier for both the patient and
the physician. This new test may decrease the need for laparoscopy
in women with pelvic pain or infertility.
SUMMARY OF THE INVENTION
[0030] The Applicant has found surprisingly that a woman with
endometriosis has a different expression of the Major
Histocompatibility Complex (MHC) class I antigens (especially
HLA-ABC surface antigens) in or on their endometrial cells, when
compared with the expression of the same antigens of endometrial
cells of a healthy woman. Possibly, this difference in the
expression of the MHC class I antigens prevents their recognition
and cytolysis by the NK (natural killer) cells. This therefore may
explain why the endometrial cells found in the peritoneal cavity
are not destroyed, following retrograde menstruation and their
subsequent peritoneal implantation. The presence of a different
expression of these antigens on endometrial cells is therefore
predictive of endometriosis.
[0031] A large number of ligands, particularly monoclonal
antibodies, that bind the aforesaid antigens normally expressed on
the surface of a large number of human cells are available. Some
authors (Johnson et al., 1984 and Sakaguchi et al., 1985) have
evaluated the antigenic expression of the HLA-ABC antigens on the
surface of endometrial cells. However the expression of the HLA-ABC
antigens has never been correlated with the presence or absence of
endometriosis. This is the first proposal suggesting that
endometrial cells from patients with endometriosis express
MHC-class I antigens, especially the HLA-ABC antigens, differently
from normal individuals or other patients.
[0032] Therefore, it is an object of the present invention to
provide the clinicians with a new application for ligands specific
to MHC-class I antigens, especially HLA-ABC antigens, this new
application residing in the detection and diagnosis of
endometriosis.
[0033] It is also an object of the present invention to provide a
test for diagnosing endometriosis, especially a non-invasive and
reliable screening test, using a ligand to these antigens for the
diagnosis of endometriosis. This antigen is present in or on
endometrial cells or cells having the same embryonic origin
(especially endocervical cells). Therefore, this method can be
practised on a specimen obtained from the endometrium of a patient
and does not require a specimen sampled directly from the
endometriotic foci. The detection of these antigens carried out
according to a process comprising the reaction of a ligand, e.g.
antibodies with the antigens normally expressed at the surface of
endometrial cells.
[0034] According to a preferred embodiment of the invention, the
test uses a ligand to an antigen originating from endometrial
cells. The MHC-class I antigens to which it will be referred
hereinbelow are defined as comprising HLA-ABC antigens and related
molecules. Such related molecules include proteic precursors like
immature proteins, portions of these antigens as well as messenger
RNAs and cDNAs. When proteins are targeted, the ligand of choice is
an antibody specific thereto, and when messenger RNAs encoding
these proteins are targeted, nucleotidic probes hybridizable
thereto might be used to detect the difference of expression of
these antigens.
[0035] MHC class I antigens, especially HLA-A, B and C antigens,
are human cell surface glycoproteins. It is generally assumed that
the Major human Histocompatibility Complex class I antigens, are
expressed by nucleated cells with some exceptions like thymus,
kidney or thyroid cells. They have a role in the interaction
between cytotoxic T-cells and target cells.
BRIEF DESCRIPTION OF THE FIGURES
[0036] The invention will be better understood upon reading the
following non limitative description of preferred embodiments of
it, reference being made to the accompanying figures in which:
[0037] FIG. 1: Effect of collagenase and trypsin on the detection
of HLA-ABC surface antigens. Peripheral blood lymphocytes (PBL)
were incubated with media and collagenase 0.25% for 2 hours at
37.degree. C., or trypsin 0.1% for 12 minutes at 37.degree. C. The
cells were labelled with a monoclonal antibody directed against
HLA-ABC surface antigens and analysed by flow cytometry (n=3),
[0038] FIG. 2: Expression of the MHC class I surface antigens on
glandular endometrial cells versus the results obtained with
laparoscopy. The cytocentrifuged endometrial cells of women were
analysed by indirect immunohistochemistry to determine the
percentage of cells expressing HLA-ABC antigens. These results were
compared with the results obtained by performing a laparoscopy on
these same women (n=60).
[0039] FIG. 3: Detection of the MHC class I surface antigens by the
kit versus the results obtained with laparoscopy. The endometrial
cells retained on chamber slide coated with a cytokeratin antibody
were analysed by indirect immunocytochemistry to determine the
percentage of cells expressing HLA-ABC antigens. These results were
compared with the results obtained by performing a laparoscopy in
these same women (n=40).
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0040] The ligands usable in the present invention are available
from different sources. Examples of ligands include antibodies
directed to MHC-class I antigens, especially antibodies directed to
HLA-ABC antigens, nucleotidic sequences, especially cDNA molecules
or oligonucleotides which are complementary to mRNAs or to cDNAs
encoding these antigens, and receptors to these antigens. Among
these ligands, the monoclonal antibody produced by the hybridoma
PHM4 has been found particularly suitable to detect the presence of
endometriosis. Of course, any monoclonal antibody having the same
or equivalent specificity as PHM4 could be used in the present
invention. This monoclonal antibody is commercialized by Chemicon
(Temecula, Calif.) as a ligand for the identification of HLA-ABC
cells. The binding of these ligands to the targeted antigens is
monitored by labelling means. These labelling means comprise but
are not limited to markers selected from the group consisting of
radioisotopes, fluorochromes, enzymes, biotin and electron dense
molecules.
[0041] The radioisotopes used for labelling may be selected from
the group consisting of 125I, .sup.131I, .sup.32P, .sup.3H,
thallium, technetium, strontium, indium and yttrium. The enzymes
used for labelling may be selected from the group consisting of
peroxidase, esterase and alkaline phosphatase.
[0042] The electron dense molecules used for labelling may be
selected from the group consisting of ferritin, gold and latex
spheres.
[0043] The endometrial cells or cell components thereof may be
evaluated on tissue section or may be suspended in an excipient
(for example RPMI-1640 medium, Roswell Park Memorial Institute) to
define a suspension. This cell suspension may be obtained by a
process comprising a mechanical disruption and/or enzymatic
treatment of endometrial tissue. The cells are mounted and fixed on
a support suitable for analysis purposes.
[0044] The step of the method mentioned hereinabove, where the
labelled antibody is being detected, may be carried out according
to a procedure comprising the step of counterstaining the cells to
reveal the presence of the endometrial cells or endometrial cell
components and detecting a resulting colouration of the antigen
with appropriate detection means.
[0045] These detection means may be selected from the group
consisting of enzyme-linked immunosorbent assay, solid phase
radiobinding immunoassays where the antibodies may be directed
either against soluble antigens or cell surface antigens,
autoradiography, competitive binding radioimmunoassay,
immunoradiometric assay (IRMA), electron microscopy, peroxidase
anti-peroxidase (PAP) labelling, fluorescent microscopy, alkaline
phosphatase labelling, peroxidase labelling, an apparatus or
material for measuring radioactivity, electrophoresis, Western
blotting, Northern blotting, Southern cDNA blotting, flow
cytometry, optical microscopy, spectrophotometry, a densitometer,
an apparatus for measuring light reactivity, an apparatus for
calorimetric determination and a polymerase chain reaction
(PCR).
[0046] In the case where the detection means used is optical
microscopy, the cells are mounted and fixed on a microscope slide.
In this case, the step of detecting the labelled antibody is
carried out according to a procedure comprising the step of
counter-staining the cells to reveal the presence of the
endometrial cells or endometrial cell components, and detecting a
resulting colouration of the antigen with an optical
microscope.
[0047] When the cells are mounted on a microscope slide, they may
be mounted by cytocentrifugation or selectively retained by
anti-cytokeratin antibodies coated on chamber slide or other
mounting techniques known in the art (e.g. smearing).
[0048] When the endometrial tissue comprising endometrial cells is
fixed on a microscope slide, it may be fixed according to a
technique selected from the group consisting of cryosection and
paraffin-embedding.
[0049] However, the cells or cell components can also be suspended
in an excipient to define a suspension and/or simply smeared on a
microscope slide for direct cytological analysis.
[0050] In the case where the cells are analysed in order to detect
the antigens by either enzyme-linked immunosorbent assay, solid
phase radiobinding immunoassays where the antibodies may be
directed either against soluble antigens or cell surface antigens,
autoradiography, competitive binding radiomimunoassay,
immunoradiometric assay (IRMA), electron microscopy, peroxidase
anti-peroxidase (PAP) labelling, fluorescent microscopy, alkaline
phosphatase labelling, peroxidase labelling, an apparatus or
material for measuring radioactivity, electrophoresis, Western
blotting, Northern blotting, Southern cDNA blotting, flow
cytometry, optical microscopy, spectrophotometry, a densitometer,
an apparatus for measuring light reactivity, an apparatus for
calorimetric determination and a polymerase chain reaction (PCR),
the endometrial cells or cell components may be suspended in an
excipient to define the suspension, isolated by a process
comprising a mechanical disruption of endometrial tissue and the
suspension is then digested.
[0051] In the case where electrophoresis is used for protein
detection, the endometrial cells or cell components may be
suspended in an excipient to define a suspension and may be
obtained by a process comprising a mechanical disruption of
endometrial tissue. Advantageously, the suspension is then digested
and deposited in a well of an electrophoresis gel. Next, the
antigen is migrated through said gel by electrophoresis so that the
antigen is isolated. Then the migrated antigens are transferred
onto a membrane (e.g. nitrocellulose, nylon). Next, either a
labelled antibody is reacted with the aforesaid membrane, or an
antibody is reacted with aforesaid membrane and reacted with the
antigen, and then a labelled antibody is further added to aforesaid
membrane, and reacted with the antibody having already reacted with
the antigen. The labelled antibody is then detected by detection
means.
[0052] The detection means mentioned hereinabove may be selected
from the group consisting of an apparatus for colorimetric
determination, an apparatus for measuring light reactivity and an
apparatus for measuring radioactivity.
[0053] Advantageously, the cells evaluated may be obtained from the
endometrium.
[0054] The endometrial cells or cell components should be glandular
endometrial cells or cell components.
EXAMPLE 1
[0055] Advantageously, as a particularly preferred direct method of
detection of HLA-ABC surface antigens of endometrial cells for the
diagnosis of endometriosis, the following steps may be carried
out:
[0056] Step 1
[0057] Cell preparation
[0058] 1.1 Endometrial biopsy with Wallach.TM. endocell
(Pharmascience) or any other endometrial sampler
[0059] 1.2 Mechanical disruption of endometrial tissue and
filtration through a 250 .mu.m stainless steel sieve
[0060] 1.3 Collect by backwash endometrial glands retained on the
filter
[0061] 1.4 Dissociate glands by incubation with trypsin 0.25% for
10 minutes at 37.degree. C. and wash 2.times. with RPMI
supplemented with 2.5% human AB (HAB) serum (Gibco, Grand Island,
N.Y.)
[0062] Step 2
[0063] Evaluation of % of cells expressing BLA-ABC
[0064] 2.1 Approximately 5.times.10.sup.5 cells obtained after step
1.4 are incubated with monoclonal antibodies including an
anti-HLA-ABC antibody (Chemicon) and a negative isotype control
antibody (Coulter, Hialeah, Fla.) for 30 minutes at 4.degree.
C.
[0065] 2.2 Wash cells twice with 2.5% HAB serum in RPMI
[0066] 2.3 Incubate with Goat anti-Mouse Ig conjugated to FITC
(Coulter) for 30 minutes at 4.degree. C.
[0067] 2.4 Wash cells twice with 2.5% HAB serum in RPMI
[0068] 2.5 Fix in 0.1% formaldehyde
[0069] 2.6 Determine immunofluorescence reactivity by flow
cytometry to determine the percentage of cells expressing
HLA-ABC
[0070] Step 3
[0071] Alternative to enhance specificity of detection of HLA-ABC
antigens
[0072] 3. In order to confirm that HLA-ABC expression is evaluated
specifically on glandular cells, it is possible to identify
glandular cells by monoclonal antibody labelling. Instead of step
2, the endometrial cells are first permeabilized and then labelled
with an anti-cytokeratin monoclonal antibody conjugated with FITC
(Becton-Dickinson, San Jose, Calif.). HLA-ABC expression is
evaluated only on cells gated for cytokeratin positivity. We have
previously shown that cell permeabilization does not impair
detection of HLA-ABC expression (FIG. 1)
[0073] 3.1 Endometrial cells obtained from step 1.4 are spun down
and supernatant removed
[0074] 3.2 Cells are put on ice and cold ethanol (-80.degree. C.)
is slowly dripped on the cells
[0075] 3.3 This cell mixture is incubated at 4.degree. C. for 30
minutes
[0076] 3.4 Cells are washed twice with 2.5% HAB serum in RPMI 1640
and divided into four tubes (approximately 5.times.10.sup.5 cells
per tube)
[0077] 3.5 Following antibodies are added to the different
tubes:
[0078] tube 1: negative MsIg (source; same isotype as anti-HLA)
unconjugated antibody
[0079] tube 2: keep at 4.degree. C. until step 3.7
[0080] tube 3: anti-HLA unconjugated antibody
[0081] tube 4: keep at 4.degree. C. until step 3.7
[0082] 3.6 Mixture is incubated at 4.degree. C. for 30 minutes and
then tubes 1 and 3 are washed twice with 2.5% HAB serum in RPMI
[0083] 3.7 Following reagents are added to the different tubes and
incubated at 4.degree. C. for 30 minutes:
[0084] tube 1: Goat anti-Mouse Ig conjugated to FITC
[0085] tube 2: negative Goat anti-MsIg (Coulter) conjugated to
FITC
[0086] tube 3: Goat anti-Mouse Ig conjugated to FITC
[0087] tube 4: anti-cytokeratin antibody conjugated to FITC
[0088] 3.8 Cells are washed twice with 2.5% HAB serum in RPMI and
fixed in 0.1% formaldehyde
[0089] 3.9 Flow-cytometry is used to determine the percentage of
cells expressing cytokeratin using standard techniques known in the
art
[0090] 3.9.1 Gating is then performed on cytokeratin positive cells
and further evaluation is done only on these gated cells
[0091] 3.9.2 Cytokeratin positive cells gated are evaluated:
Expression of HLA-ABC is then evaluated using cells from tube 4 and
negatively labelled cells from tube 2 are used as negative
controls
[0092] 3.9.3 This assay allows determination of the percentage of
endometrial cells expressing HLA-ABC
EXAMPLE 2
[0093] Also, as another particularly preferred method of detection
of HLA-ABC surface antigens of endometrial cells for the diagnosis
of endometriosis, the following steps may be carried out:
[0094] Step 1
[0095] Cell preparation
[0096] 1.1 Endometrial biopsy with Wallach.TM. endocell
(Pharmascience) or any other endometrial sampler
[0097] 1.2 Tissue section by cryosection and then mounted on
slide
[0098] Step 2
[0099] Evaluation of % of cells expressing HLA-ABC
[0100] 2.1 Proceed to fixation with acetone/methanol (3:1) for 6
minutes
[0101] 2.2 Wash with Tris-HCL buffer for 5 minutes
[0102] 2.3 Incubation with blocking agent (BSA 0.5%) for 5
minutes
[0103] 2.4 Add anti-HLA-ABC antibody and incubate for 30
minutes
[0104] 2.5 Wash with Tris-HCL buffer 3.times. for 1 minute
[0105] 2.6 Incubate with second antibody biotinylated (Dako,
Pittsburg, Pa.) for 10 minutes
[0106] 2.7 Wash with Tris-HCL buffer 3.times. for 1 minute
[0107] 2.8 Incubate with enzyme-conjugated streptavidin for 10
minutes
[0108] 2.9 Wash with Tris-HCL buffer 3.times. for 1 minute
[0109] 2.10 Incubate with substrate for 10 minutes
[0110] 2.11 Wash 1.times. with tap water for 1 minute
[0111] 2.12 Counter-stain with Mayer's heamalun for 1 minute
[0112] 2.13 Wash in tap water for 5 minutes
[0113] 2.14 Mount slide with aqueous solution and read under
optical microscope to determine the percentage of cells expressing
HLA-ABC
EXAMPLE 3
[0114] Furthermore, as still another particularly preferred method
of detection of MHC Class I surface antigens in endometrial cells
by electrophoresis for the diagnosis of endometriosis, the
following steps may be carried out:
[0115] Step 1
[0116] Cell preparation
[0117] 1.1 Endometrial biopsy with Wallach.TM. endocell
(Pharmascience) or any other endometrial sampler
[0118] 1.2 Mechanical disruption of endometrial tissue and
filtration through a 250 .mu.M stainless steel sieve
[0119] 1.3 Collect by backwash endometrial glands retained on the
filter
[0120] 1.4 Dissociation of glands by incubation with trypsin 0.25%
for 10 minutes at 37.degree. C. and wash 2.times. with RPMI
supplemented with 2.5% FCS
[0121] Step 2
[0122] Gel technique
[0123] 2.1 Preparation of 1% SDS-12% polyacrylamide gel
[0124] 2.2 Mix cell suspension with equal volume of loading
buffer
[0125] 2.3 Place the sample in boiling-water for 10 minutes
[0126] 2.4 Deposit mixed suspension in wells and run in
conventional manner the electrophoresis gel
[0127] 2.5 When the SDS-polyacrylamide gel is approaching the end
of its run, rinse the graphite plate with distilled water and wipe
off any beads of liquid that adhere to them with Kimwipes.TM.
[0128] 2.6 Cut six pieces of Whatman.TM. 3MM paper and one piece of
nitrocellulose filter (Millipore) to exact size of the
SDS-polyacrylamide gel
[0129] 2.7 Float the nitrocellulose filter on the surface of a tray
of deionized water and allow it to wet from beneath by capillary
action. Then submerge the filter in the water for at least 5
minutes
[0130] 2.8 Soak the six pieces of Whatman.TM. 3MM paper in a
shallow tray containing a small amount of transfer buffer
[0131] 2.9 Set up the transfer apparatus
[0132] 2.10 Place the upper electrode on top of the stack, graphite
side down. Connect the electrical leads and apply a current of 0.65
mA/sq. cm of gel for a period of 1.5-2.0 hours
[0133] 2.11 Disassemble the transfer apparatus from the top
downward, peeling off each layer in turn. Transfer the gel to a
tray containing Coomassie Brillant Blue and stain it
[0134] 2.12 Cut off the bottom left-hand corner of the filter and
stain the filter with a radiolabelled antibody or other suitable
probe against HLA-ABC surface antigens and visualize by
autoradiography to detect said surface antigens
[0135] The examples which follow provide an illustration of the
characteristics and advantages of the present invention, without
however limiting its scope.
EXAMPLE 4
Method of Obtaining Endometrial Tissue Samples
[0136] The endometrial tissue samples were obtained from 60 women
undergoing diagnostic laparoscopy, from infertile patients
attending a fertility clinic, from women undergoing procedures
related to gynaecological pathologies or from women undergoing
tubal ligation. These samples were taken with a Wallach.TM.
endocell sampler (Pharmascience, Montreal, Qubec), during the
secretory phase of the menstrual cycle (between the 18th day and
the 25th day of the cycle). The samples were placed into sterile
RPMI-1640 (Roswell Park Memorial Institute) medium (Gibco, Grand
Island, N.Y.) and supplemented with 10% heat-inactivated Fetal Calf
Serum (Gibco, Grand Island, N.Y.), 2% L-glutamine and 1%
penicillin/streptomycin. Of course, the samples may have been
placed in other culture media or aqueous suspensions suitable to
maintain cell viability.
Isolation of a Glandular Endometrial Cell
[0137] The endometrial tissue was suspended in an excipient
(RPMI-1640) and mechanically disrupted using Pyrex.TM. glass
Broeck.TM. tissue grinders (Fisher, Nepean, Ont.) in order to
obtain an endometrial cell or cell component suspension. The
alternative excipient may be selected amongst well known media.
[0138] The suspension was then filtered through a 250 .mu.M
stainless steel sieve (Millipore, Marlborough, Mass.), where the
glands were retained on the filter, while the stromal enriched
fraction passed through the filter. These glands were recovered
from the filter by backwashing with RPMI medium.
[0139] The glandular cells or cell components were then incubated
with collagenase type I 0.25% (Sigma, St-Louis, Mo.) for about 2
hours at 37.degree. C., in a shaking waterbath. Subsequently, the
glandular cells or cell components were incubated with trypsin 0.1%
(Sigma, St-Louis, Mo.) for about twelve minutes at 37.degree. C.
The enzymatic reaction was stopped with 500 .mu.L of normal human
serum. After treatment with trypsin 0.1%, the glandular endometrial
cell was washed twice with RPMI medium.
[0140] Another method (Satyaswaroop, 1979) may be used to isolate a
glandular endometrial cell. Here, the endometrial tissue is cut
into about 1 mm. pieces and treated with 0.25% collagenase at
37.degree. C., in a shaking water bath for two hours. This
collagenase-treated tissue is then strained through a 250 .mu.M
sieve in order to retain the undigested tissue and mucous material.
The filtrate is then passed through a 38 .mu.M or 105 .mu.M sieve
in order to retain the glands. In order to disperse the glands into
single glandular epithelial cells, it is possible to add a second
incubation period. The glands were then resuspended in 3 mL of
trypsin 0.025%-EDTA 0.01% and in PBS/BSA, and then incubated for
seven to twelve minutes. However, there are disadvantages with this
technique in that firstly, one must carry out additional
filtrations on 38 .mu.M or 105 .mu.M sieves to obtain these glands
and secondly, because the purity after the second filtration would
not be as good as that for the initial method.
[0141] The endometrial tissue can also be evaluated on a slide
without being in suspension, using the techniques of cryosection or
paraffin-embedding of tissues.
Cytocentrifugation
[0142] The endometrial cells were taken at different steps of the
isolation procedure or sorted using flow cytometry, evaluated for
viability and then cytocentrifuged (Shandon Cytospin II,
Pittsburgh, Pa.) and dried on a slide for thirty minutes. The slide
was then stained with Papanicolaou stain (Sigma, St-Louis, Mo.) for
morphological confirmation, or with various monoclonal
antibodies.
[0143] With regard to the evaluation for viability, it is to be
noted that the glandular cells were well dispersed and conserved
their integrity. In order to evaluate the purity of the glandular
fraction, the glandular cells were measured with a monoclonal
antibody (mAB) directed against the cytokeratin 14-18 antibody and
visualized by alkaline phosphatase staining. The glandular fraction
was contaminated by less than 25% of cells which were negative for
the presence of cytokeratin 14-18 (Table I).
1 TABLE I DETECTION ON DETECTION BY CYTOCENTRIFUGED FLOW CELLS
CYTOMETRY % PURITY 75 96 % HLA-ABC WITHOUT 31 .+-. 24 22 .+-. 11
ENDOMETRIOSIS % HLA-ABC WITH 90 .+-. 10 79 .+-. 11
ENDOMETRIOSIS
[0144] The contamination was made by the presence of the stromal
cells (i.e. fibroblast-derived cells and leucocytes). Other
isolation techniques can also be used which give similar
results.
[0145] The cytocentrifuged cells were fixed on the microscope slide
with a mixture of acetone/methanol (3:1) for ten (10) minutes and
washed 2 times with Tris-HCl buffer for five minutes. Optionally,
the cells may be washed 2 or 3 times with Tris-HCl buffer for 1 to
5 minutes. The cells can be fixed using other well known fixation
techniques known in the art.
[0146] A blocking reagent, BSA 0.1%, was then added for five
minutes to saturate non-specific binding sites. Powdered milk may
also be used as a blocking reagent. Optionally, the blocking
reagent, BSA 0.1%, may be added for five to thirty minutes.
Addition of a Monoclonal Antibody By Indirect
Immunohistochemistry
[0147] The slide was incubated with a primary mouse monoclonal
antibody for thirty minutes at room temperature and washed three
times with Tris-HCl buffer for five minutes each time. The antibody
selected was an anti-HLA-ABC (Chemicon, Temecula, Calif.). The
slide was reincubated with an anti-mouse
Immunoglobulin-biotinilated monoclonal antibody for 10 minutes at
room temperature, and washed three times with Tris-HCl buffer for
five minutes each time. Optionally, the incubation times for both
monoclonal antibodies may vary from 30 minutes to 1 hour at a
temperature varying from 4.degree. C. to room temperature.
[0148] Optionally, the slide may be washed between each exposure to
antibody, 2 or 3 times with Tris-HCl buffer for 10 1 to 5
minutes.
[0149] Streptavidine conjugated with alkaline phosphatase was then
added to form a complex with biotin already associated with the
anti-mouse Immunoglobulin monoclonal antibody, for ten minutes and
the slide was again washed three times with Tris-HCl buffer for
five minutes each time. Optionally, the slide may be washed 2 or 3
times with Tris-HCl buffer for 1 to 5 minutes. The slide was
developed with the addition of a chromogenic substrate for ten
minutes, and washed with tap water for one minute. The cytospin
slide was counterstained with Mayer's haemalun (BDH, Toronto, Ont.)
for one minute and washed with tap water for another five minutes
Other stains known in the art like Wright-Giemsa may be used to
counterstain the slides. Finally, the slide was mounted with
Immu-mount (Shandon, Pittsburgh, Pa.). The staining was evaluated
by optical microscopy in at least 3 times 100 cells. When the
endometrial cells express the HLA-ABC surface antigen, these cells
have their surface antigens revealed by a characteristic
colouration.
[0150] FIG. 2 shows the expression of HLA-ABC antigens at the
surface of glandular endometrial cells of women suffering or not of
endometriosis versus the diagnosis obtained by performing
laparoscopies on these women. The laparoscopy results showed that
for 60 women tested, 35 did not have endometriosis while 25 did
have endometriosis. However, using our indirect immunohistochemical
method, 7 of the 35 women who did not have endometriosis by
laparoscopy, showed a high percentage (specificity >80%) of
endometrial cells expressing HLA-ABC antigens with a different
expression, which is predictive of endometriosis. Therefore, the
results obtained by laparoscopy suggest that they could be less
reliable than those with our indirect immunohistochemical method
which showed a 100% sensitivity. For example, it has been
demonstrated that false negative results with laparoscopy could
occur in women with microscopic endometriosis. In our indirect
immunohistochemical method, women without endometriosis expressed
only 31.+-.24% (mean.+-.S.D.) while women with endometriosis
expressed 90.+-.10% HLA-ABC antigens. Class II Major
Histocompatibility Complex antigens were not expressed at the
surface of glandular endometrial cells from women with or without
endometriosis (data not shown). It should be deduced from the
results shown in FIG. 2 that all women with laparoscopically
diagnosed endometriosis have at least about 65% of their
endometrial cells expressing HLA-ABC antigens. In the presence of
negative results (less than 65%) in our immunohistochemical
diagnostic test, endometriosis would be ruled out, preventing the
need for a laparoscopy in these women. However, in the presence of
positive results (.gtoreq.65%) in our immunohistochemical
diagnostic test, laparoscopy could still be necessary in these
women to evaluate the invasiveness (stage 0 to IV) of
endometriosis.
[0151] It was further verified that enzymatic steps in the
isolation method mentioned hereinabove, do not cleave the epitope
of antigenic structures found on a cell surface. In order to verify
this, normal peripheral blood mononuclear cells were treated with
collagenase and trypsin, as they are used for this isolation
procedure, in order to evaluate whether or not enzymatic treatment
has an effect on the expression of surface antigens of peripheral
blood lymphocytes (PBL) from normal donors.
[0152] Indeed, antigenic structures could be cleaved by enzymatic
treatment resulting in a modification of the pattern of expression
of different surface antigens, for example leading to the absence
of detection of an antigen that is really present on native cells
but cleaved by the enzymatic treatment.
[0153] PBL were incubated with collagenase type I 0.25% (Sigma,
St-Louis, Mo.) for 2 hours at 37.degree. C. in a shaking waterbath
and washed twice and with trypsin 0.1% (Sigma, St-Louis, Mo.) for
12 minutes at 37.degree. C. Enzymatic reaction was stopped with 500
.mu.L of normal human serum. As control, PBL were incubated with
medium only (no enzymatic treatment). Treated and untreated cells
were then labelled with monoclonal antibodies for
immunofluorescence analysis.
[0154] As shown in FIG. 1, treatment with collagenase or with
trypsin did not affect the recognition of HLA-ABC surface antigens
on PBL. Although this immunological evaluation was performed on
peripheral blood cells, it is possible to extrapolate that the
enzymatic treatment will not affect recognition of HLA-ABC surface
antigens on glandular endometrial cells.
EXAMPLE 5
Flow Cytometry
[0155] All the procedures set forth in step 1 of Example 1 were
repeated until the addition of the monoclonal antibody. This
addition is then carried out according to the following steps:
[0156] A panel of monoclonal antibodies was used. The cytokeratin
conjugated antibody with FITC (Becton Dickinson, San Jose, Calif.)
and an unconjugated HLA-ABC were used. Labelling of cells with this
panel of monoclonal antibodies was performed using standard
techniques known in the art.
[0157] Briefly 0.5 times 10.sup.6 cells were incubated with
monoclonal antibodies for 30 minutes at 4.degree. C., then washed
twice. A second incubation was performed for the unconjugated
antibody with anti-MsIg conjugated to phycoerythin (PE) for 30
minutes at 4.degree. C., then washed twice and fixed in 0.1%
formaldehyde. Immunofluorescence reactivity was determined by flow
cytometry analysing 10.sup.4 cells in each sample. Flow-cytometry
(FACstar plus, Becton-Dickinson, San Jose, Calif.) was performed
with an argon laser operating at 488 nm with an intensity of 200
mW. Background fluorescence was determined using isotype-matched
nonreactive directly or indirectly conjugated monoclonal antibodies
(MsIg-FITC and PE) and was <1% for all analysed. Table I shows
the results for 12 samples analysed by flow cytometry. Women
without endometriosis expressed only 22.+-.11%, in contrast with
women with endometriosis who expressed 79.+-.11% HLA-ABC
antigens.
EXAMPLE 6
Method of Obtaining Endometrial Tissue Samples
[0158] The endometrial tissue samples were obtained from 40 women
undergoing diagnostic laparoscopy, from infertile patients
attending a fertility clinic, from women undergoing procedures
related to gynaecological pathologies or from women under tubal
ligation. These samples were taken with a Wallach.TM. endocell
sampler (Pharmascience, Montral, Qubec), during the secretory phase
of the menstrual cycle (between the 18th day and the 25th day of
the cycle). The samples were placed into sterile RPMI-1640 medium
and supplemented with 10% heat-inactivated Fetal Calf Serum, 2%
L-glutamine and 1% penicillin/streptomycin. Of course, the samples
may have been placed in other culture media or aqueous suspensions
suitable to maintain cell viability.
Tissue Preparation
[0159] The endometrial tissue was cut in small pieces (3 mn.times.3
mn) with 2 scalpels and placed in a round bottom test tube. In
order to generate a cell suspension, the tissue was digested with
an enzymatic solution containing collagenase type I 0.4% and DNAse
type I 0.001% for 45 minutes at 37.degree. C. in a shaking
waterbath. After the treatment, the cell suspension was washed
twice with RPMI medium.
[0160] The cell must be permeabilized before the evaluation
procedure. The endometrial cells are spun down and supernatant
removed. Add drop by drop, 1 mL of acetone/methanol (3:1) on the
pellet and gently resuspend the cells. Incubate 6 minutes at room
temperature to achieve permeabilization. Add slowly 9 mL of water
and spin down.
Pre-Coated Chamber Slide
[0161] The evaluation of the MHC class I antigen is specific only
on the endometrial glandular cells. The cell type selection is
achieved using the anti-cytokeratin antibody specific for
epithelial cells. A solid support like the Nunc chamber slide (4, 8
or 16 chambers can be used) was pre-coated with a rabbit
anti-human-cytokeratin pan (15 .mu.g/mL) and the cell suspension
put in the chambers. After a 15-minute incubation at room
temperature, the chambers were washed twice with Tris-HCl
buffer.
[0162] A blocking reagent, BSA 2% was then added for five minutes
to saturate non-specific binding sites. These pre-coated chamber
slides comprising fixed anti-cytokeratin antibody are usable to
retain the endometrial glandular cells.
Addition of a Monoclonal Antibody By Indirect
Immunocytochemistry
[0163] The chamber slides were incubated with a primary mouse
monoclonal antibody for 15 minutes at room temperature and washed
three times with Tris-HCl buffer for 1 minute each time. The
antibody selected was an anti-HLA-ABC (Chemicon, Temecula, Calif.).
The chambers slide were reincubated with an anti-mouse
immunoglobulin-biotinylated polyclonal antibody for 10 minutes at
room temperature, and washed three times with Tris-HCl buffer for 1
minute each time.
[0164] Streptavidine conjugated with alkaline phosphatase was then
added to form a complex with biotin already associated with the
anti-mouse immunoglobulin monoclonal antibody, for 10 minutes and
the chamber slide were again washed three times with Tris-HCl
buffer for 1 minute each time. The fixed primary antibody was
developed with the addition of a chromogenic substrate for 10
minutes, and washed with tap water for 1 minute. The cells were
counterstained with Mayer's haemalun for 45 seconds and washed for
another 5 minutes. The chambers were taken off from the slide.
Finally, the slide was mounted with immu-mount (Shandon,
Pittsburgh, Pa.). The staining was evaluated by optical microscopy
in at least 3 fields of 100 cells. When the endometrial cells
express the HLA-ABC surface antigen, these cells have their surface
antigens revealed by a characteristic colouration.
[0165] FIG. 3 shows the expression HLA-ABC antigens at the surface
of glandular endometrial cells of women suffering or not of
endometriosis versus the diagnosis obtained by performing
laparoscopies on these women. The laparoscopy results showed that
for 40 women tested, 24 did not have endometriosis while 16 did
have endometriosis. However, using our indirect immunocytochemical
method, 6 of the 24 women who did not have endometriosis by
laparoscopy, showed a high percentage of endometrial cells
expressing HLA-ABC antigens with a different expression, which is
predictive of endometriosis. Therefore, the results obtained by
laparoscopy suggest that they could be less reliable than those
with our indirect immunocytochemical method which showed a 100%
sensitivity. For example, it has been demonstrated that the false
negative results with laparoscopy could occur in women with
microscopic endometriosis. In our indirect immunocytochemical
method, women without endometriosis expressed only 37.+-.34%
(mean.+-.SD) while women with endometriosis expressed 89.+-.6%
HLA-ABC antigens. It may be deduced from the results shown in FIG.
3 that all women with laparoscopically diagnosed endometriosis have
more than 65% of their endometrial cells expressing HLA-ABC
antigens. In the presence of negative results (<65%) with
immunocytochemical diagnostic test, endometriosis would be ruled
out, preventing the need for a laparoscopy in these women. However,
in the presence of positive results (>65%) with
immunocytochemical diagnostic test, laparoscopy could still be
necessary in order to evaluate the invasiveness of
endometriosis.
[0166] It appears clearly from the above results that the present
invention provides a method which will easily, rapidly and
sensitively differentially detect endometriosis in women when
compared to those not suffering from the same.
Diagnostic Kit
[0167] The method of diagnosing endometriosis according to this
invention can be practised by way of a diagnostic kit. Such a kit
should contain all the essential elements to perform one of the
preferred methods, which choice depends on the laboratory
facilities available to the practician. Such essential elements are
the following for detecting the presence of antigens:
[0168] Indirect detection:
[0169] a first antibody consisting of an anti-MHC class I antibody,
preferably a monoclonal anti-HLA-ABC antibody, and
[0170] a labelled second antibody which binds antibodies all the
antibodies of the first species.
[0171] Direct detection:
[0172] a labelled anti-MHC class I antibody, preferably a
monoclonal anti-HLA-ABC antibody.
[0173] Optional reagents and material:
[0174] When the method involves disruption of the sample cells as
in Example 1, reactants like trypsin, RPMI and human AB serum may
optionally enter the composition of the kit as well as any buffer
or reagent for the revelation of the binding of the antibodies to
the antigens and the disposable instruments and reagents for
sampling endometrium. Optionally, a labelled appropriately selected
anti-cytokeratin antibody may be provided.
[0175] When the method involves the mounting of a biopsy on a slide
as in Example 2, the same optional components may enter the
composition of the test kit except for the material and buffer for
disrupting the cells.
[0176] When the method involves the separation of proteins by
electrophoresis as in Example 3, no human AB serum is necessary.
Also, the material and reagents necessary for the electrophoresis
and subsequent blotting may be added.
[0177] Finally, when the method involves tissue separation and
retention of glandular endometrial cells as in Example 6, an
enzymatic solution for separation and anti-cytokeratin pre-coated
chamber slides for retention may be provided along with the
reagents necessary to reveal the binding of the cells to anti-MHC
class I antibody.
* * * * *