The level of EF in the sample can be assayed using, for example, a lymphocyte/platelet binding assay. The invention is based on the discovery that serum derived from subjects having endometriosis contains elevated levels of a factor that decreases the level of of preimplantation factor (PIF) activity found within the serum of pregnant females. PIF activity can be detected by its ability to increase the binding of platelets to lymphocytes. However, in the presence of EF, PIF mediated platelet/ lymphocyte binding is decreased. The present invention further provides for kits for carrying out the above described screening methods. Such kits will be used to screen subjects for increased levels of EF, as a diagnostic, prognostic or predictive indicator of endometriosis. The invention is demonstrated by way of examples in which increased levels of EF were identified in serum samples derived from subjects with endometriosis.

2. BACKGROUND OF THE INVENTION Endometriosis is a disease in which endometrial tissue, normally localized to the uterine lining, is found outside the uterus. It is believed that during the menstrual period, endometrial cells scale off, are transported through the fallopian tubes into the peritoneal cavity where they implant, proliferate and develop into endometriotic lesions (Sampson, J. A., 1927, Am. J. Obstet Gynecol. 14: 422-469).

This displaced endometrial tissue is normally confined to the abdominal cavity and

commonly adheres to the ovaries and ligaments that support the uterus. Since the displaced endometrial tissue responds to the same hormones that the uterus responds to, the displaced tissue may bleed during the menstrual period and may cause cramps, pain and result in the formation of scar tissue. Endometriosis is commonly diagnosed among women undergoing evaluation for infertility and it is believed that 25 to 50 percent of infertile women may have endometriosis resulting in physical blocking of the ovaries.

Data suggests that in normal situations, immunological clearance is responsible for protection against growth of endometrial cells in ectopic sites (Vigano, P. et al., 1991, Fertil Steril. 56: 894-899; Oosterlynck, D. J., et al., 1991, Fertil Steril.

56: 45-51; Oosterlynck, D. J., et al., 1991, Fertil Steril. 58: 290-295; Ho, H. N., et al., 1995 Hum Reprod. 10: 2671-2675; Vigano, P. et al., 1994, Am J. Reprod. Immunol 32: 139-145). An alteration in immune recognition and killing of misplaced endometrial cells has been suggested as a cause for the initiation and progression of endometriosis. In particular, a role for natural killer (NK) cells as effectors of peritoneal immune surveillance has been proposed. Interleukin-12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, is known to have a potent regulatory effect on NK cells. Mazzeo et al., (1998, J Clin Endocrinolog Metab.

83: 911-916) have demonstrated that p40 levels are higher in peritoneal fluid of patients with endometriosis compared to those in women without the disease. The observed excess of p40 present in the peritoneal fluid of patients with endometriosis suggest that a NK cell defect may play a role in development of the disease.

In addition, advanced endometriosis has been demonstrated to be associated with a decrease in peripheral blood polymorphonuclear leukocyte chemotactic index and a decrease in natural killer cytotoxicity. A significant inverse correlation was also observed between plasma prostaglandin and estradiol levels and the observed decrease in chemotactic index and natural killer cytotoxicity. (Garzetti, GG et al., 1998, Obstetrics & Gynecology 91: 25-29). Thus, current data supports an association between endometriosis and alterations in immune responses which mediate the release of growth factors, prostaglandins, complement components, and

lymphokines with a subsequent effect on monocyte-macrophage activity and natural killer cell cytotoxicity.

Currently, endometriosis is diagnosed using a variety of different procedures such as ultrasound, computer tomography (CT) and magnetic resonance imaging (MRI). In some cases laparoscopy may be performed by the physician and may include a biopsy of the tissue. Certain blood tests that detect markers for endometriosis such as CA-125 and antibodies to endometrial tissue may also be used to diagnose endometriosis in a subject. However, these markers are also increased in other diseases, thereby rendering them unreliable markers of endometriosis.

It has recently been reported that a protein referred to as endometriosis protein-1 (ENDO-1) is secreted by endometriotic lesions. Amino acid sequence <BR> <BR> <BR> <BR> analysis of ENDO-1 indicates that the protein shares significant homology with the ß- chain of rat, mouse and human haptoglobin protein (Hp). In humans, distinct subtypes of Hp as well as proteins sharing epitopes with Hp have been used to diagnose a variety of different diseases such as diabetes, Alzheimer's disease and breast and prostate cancer. Increased expression of ENDO-1 in endometriotic lesions may provide a non-surgical diagnostic tool for endometriosis (Sharpe-Timms, K. L., 1991, Biology of Reproduction 58: 988-994).

Lymphocyte/platelet binding assays have been used to detect a number of different factors in the serum of subjects. For example, lymphocyte/platelet binding assays have been used to detect preimplantation factor (PIF) in the serum of pregnant females as described in U. S. Patent No. 5,645,003. The presence of PIF was shown to be a positive indicator of pregnancy and was found to be useful in predicting the spontaneous loss of pregnancies. Furthermore, it was proposed in U. S. Patent No.

5,645,003 that assays for PIF activity could be used as a diagnostic tool for endometriosis. Such an assay is based on PIF mediated enhancement of lymphocyte/platelet binding activity. This is in contrast to the present invention which provides methods for diagnosing endometriosis in a subject based on EF mediated reduction in binding of lymphocytes to platelets.

3. SUMMARY OF THE INVENTION It is an object of the present invention to provide methods for the diagnostic and prognostic evaluation of subjects having endometriosis, and for the identification of subjects possessing a predisposition to endometriosis, based on the detection of increased levels of EF in biological fluid samples of the subjects. Such information may be useful in diagnosing and treating infertility. The invention comprises assays developed to detect the level of EF in a subject's serum sample using assays such as lymphocyte/platelet binding assays.

Isolation and identification of proteins constituting EF, made possible by the discovery that increased levels of EF may be detected using lymphocyte/platelet binding assays, permits their application to development of diagnostic and therapeutic tools. Such tools include antibodies that bind to EF and which can be used in immunoassays for detection of EF levels in samples of subjects.

The invention further provides for pre-packaged diagnostic kits which may be conveniently used in clinical settings, to diagnose patients having endometriosis or a predisposition to developing endometriosis.

In a specific working example, increased levels of EF were detected in serum samples derived from subjects with endometriosis. The finding that levels of EF are increased in the serum of endometriosis subjects provides a basis for development of diagnostic and prognostic methods as well as a means for monitoring the efficacy of various therapeutic treatments for endometriosis.

4. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A, 1B. Dose Dependent Inhibitory Effect of Endometriosis Sera on PIFActivity. Addition of increasing levels of endometriosis sera, compared to controls, led to a decrease in PIF activity. The first two samples are positive and negative controls respectively in panel A and B. The endometriosis sample alone was negative for PIF activity. As indicated in panel, when more than 6p1 of negative sera was added to the control the assay became negative.

Figure 2. Dose Dependent Inhibitory Effect of Endometriosis Sera on PIF Activity. Addition of positive pregnancy sera led to an increase in PIF activity while addition of endometriosis sera to PIF positive sera led to sequential decrease in PIF activity.

Figure 3. Dose Dependence of Endometriosis Fluid for Inhibition of Lymphocyte/platelet Binding. Increasing doses of peritoneal fluid caused a decrease in lymphocyte/platelet binding compared to controls.

Figure 4. Analytical Gel Filtration of Endometriosis Associated Factors From Sample #1. Gel permeation chromatography (GPC) was conducted using a Shodex KW-803 column. Collected fractions were lyophilized and assayed for EF activity.

Figure 5. Gel Filtration HPLC Separation of Endometriosis Associated Factors From Sample #14. Gel permeation chromatography (GPC) was conducted using a Shodex KW-803 column. Collected fractions were lyophilized and assayed for EF activity.

Figure 6. Detection of EF in HPLC Collected Fractions Using Lymphocyte/Platelet Binding Assays. Known endometriosis samples from two patients (patient #1 and #14) were separated on a gel filtration HPLC column and individual samples were collected. The samples were lyophilized and resuspended in a small volume and tested in lymphocyte/platelet binding assays. Results are presented as number of cells with three or more bound platelets. EF activity was observed in the same fractions in both samples.

Figure 7. Reversed Phase HPLC of Sample #1. The fractions determined to contain EF activity were pooled and injected onto a C5 HPLC column. One ml fractions were collected, the samples were dried, resuspended in distilled water and assayed for EF activity.

Figure 8. Reversed Phase HPLC of Sample #14. The fractions determined to contain EF activity were pooled and injected onto a C5 HPLC column. One ml fractions were collected, the samples were dried, resuspended in distilled water and assayed for EF activity.

Figure 9. Detection of EF in Reverse Phase HPLC Collected Fractions Using Lymphocyte/Platelet Binding Assays. Known endometriosis samples from two

patients (patient #1 and #14) were separated on a reverse phase gel filtration HPLC and individual samples were collected. The samples were lyophilized and resuspended in a small volume and tested in lymphocyte/platelet binding assays.

Results are presented as the number of cells with three or more bound platelets. EF activity was observed in the same fractions in both samples.

5. DETAILED DESCRIPTION OF THE INVENTION The present invention achieves a highly desirable objective, namely providing methods for the diagnostic and prognostic evaluation of subjects with endometriosis and the identification of subjects exhibiting a predisposition to developing endometriosis. The assays of the invention comprise methods designed to detect increased levels of EF in serum or other biological fluids of a subject.

Specifically, the invention encompasses a method for diagnosis and prognosis of endometriosis in a subject comprising: (a) detecting endometriosis factor (EF) in a biological fluid sample derived from a subject; and (b) comparing the level of EF detected in the subject's sample to the level of EF detected in a control sample, wherein an increase in the level of EF detected in the subject's sample as compared to control samples is an indicator of the presence of endometriosis or at increased risk for endometriosis.

A wide variety of subject samples that may contain EF (s) can be prepared or assayed for the levels of EF. In preferred non-limiting embodiments of the invention, biological fluids such as urine, saliva, cerebrospinal fluid, synovial fluid, peritoneal fluid, pleural effusion, pericardial effusion, menstrual fluids, tears or sera in which secreted proteins are localized can be used to screen for increased levels of EF expression.

The present invention also provides for kits for carrying out the above- described methods. The methods can be performed, for example, by utilizing pre- packaged diagnostic kits comprising at least one reagent for detecting EF, such as an anti-EF antibody. Alternatively, the diagnostic kits comprise components for

performing lymphocyte/platelet binding assays. The present invention is based on the discovery that levels of EF in serum, as measured by inhibition of PIF activity, are increased in subjects with endometriosis.

5.1. ASSAYS FOR DETECTION OF EF EXPRESSION In accordance with the invention, measurement of levels of EF in serum or body fluids can be used for the early diagnosis of endometriosis. Moreover, the monitoring of EF levels can be used prognostically to stage the progression of the disease and to evaluate the efficacy of compounds used to treat endometriosis. In yet another embodiment of the invention, measurement of EF levels in serum or body fluids can be used in the diagnosis and evaluation of infertility.

The detection of EF in a body fluid from a subject can be accomplished by any of a number of methods. Preferred diagnostic methods for the detection of EF in a body fluid from a subject can involve, for example, measuring the inhibition of PIF activity using a lymphocyte/platelet binding assay. Such an assay for EF in the body fluid of a subject comprises the following steps: (a) removal of cells, cell debris and tissue elements from a body fluid sample of a subject; (b) providing blood lymphocytes containing platelets; (c) providing PIF activity; (d) providing an anti-CD2 antibody; (e) admixing said body fluid, lymphocytes containing platelets, PIF activity and anti-CD2 antibody; and (f) determining in the admixture the percentage of lymphocytes bound to platelets; whereby a percentage significantly lower than the percentage in a normal control sample or in the foregoing mixture lacking the body fluid indicates the presence of EF in the serum. As indicated in Figure 1, the percentage of lymphocyte binding decreases from 18% to less than 12 % depending on the amount of endometriosis serum added. Therefore, a test is considered positive when the percentage of lymphocytes bound to platelets is less than 12%.

The detection of EF expression can also be used to monitor the efficacy of potential anti-endometriosis compounds during treatment. For example, the level of EF expression can be determined before and during treatment. The efficacy of the compound can be followed by comparing EF expression throughout the treatment.

Compounds exhibiting efficacy are those which decrease the level of EF expression as treatment with the compound progresses.

The present invention is demonstrated by way of example wherein elevated levels of EF was detected in serum samples derived from endometriosis subjects. The detection and/or quantitative measurement of EF in serum or other body fluids can be used in screening of subjects who are at risk for developing endometriosis or who are being evaluated for infertility.

5.2. PREPARATION OF EF ANTIBODIES Native EF protein can be purified from natural sources, by known protein purification techniques including chromatography (e. g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility or by any other standard technique for the purification of proteins. Lymphocyte/platelet binding assays may be used to detect the presence of EF throughout the purification protocol.

According to the invention, EF protein, its fragments or other derivatives, or analogs thereof, may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a specific embodiment, antibodies to the EF protein are produced. In another embodiment, antibodies to a domain of the EF protein are produced.

Alternatively, it may not be necessary to purify EF protein for production of antibodies to the EF protein. For example, a sample containing a mixture of proteins, including EF, may be used as an immunogen to generate antibodies that immunospecifically bind to EF. The antibodies may be screened based on their ability to inhibit EF activity as determined using a lymphocyte/platelet binding assay.

Various procedures known in the art may be used for the production of polyclonal antibodies to a EF protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of a EF protein are obtained.

For the production of antibody, various host animals are immunized by injection with the native EF protein, or a synthetic version, or derivative (e. g., fragment or conjugate) thereof, including but not limited to rabbits, mice, rats, goats, etc..

Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a EF protein sequence or analog thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256: 495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Endometriosis Therapy, Alan R. Liss, Inc., pp. 77-96) may be used to produce monoclonal antibodies.

In the production of antibodies, screening for the desired antibody is accomplished by techniques known in the art, e. g. ELISA (enzyme-linked immunosorbent assay). Antibodies may be screened directly for their ability to bind to EF protein. Alternatively, antibodies which recognize a specific domain of a EF protein and/or inhibit EF activity may be selected using a lymphocyte/platelet binding assay. Using such an assay, one assays hybridomas for a product which specifically binds to EF and inhibits the EF mediated decrease in binding of lymphocytes to platelets.

5.1.3. EF IMMUNOASSAYS Immunoassays wherein EF is detected by its interaction with a EF specific antibody may be used in diagnostic methods. Antibodies useful in the present invention may be used to quantitatively detect the presence of EF in the biological fluid samples of subjects suspected of having endometriosis.

Immunoassays to be used in the practice of the invention include but are not limited to assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),"sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.

A biological sample which may contain EF, such as serum or other biological fluids in which secreted proteins can localize, is obtained from a subject suspected of having endometriosis or at risk for developing endometriosis.

Immunoassays for detection of EF typically comprise contacting the biological sample, such as a serum sample derived from a subject, with an anti-EF antibody under conditions such that specific antigen-antibody binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody. In a specific aspect, such binding of antibody, for example, can be used to detect the presence and increased expression of EF wherein the detection of increased expression of EF is an indication of a diseased condition. The levels of EF in a serum sample are compared to norms established for normal individuals without endometriosis.

In an embodiment of the invention, the biological sample, such as a serum sample is brought in contact with a solid phase support or carrier, such as nitrocellulose, for the purpose of immobilizing any proteins present in the sample.

The support is then washed with suitable buffers followed by treatment with detectably labeled EF specific antibody. The solid phase support is then washed with the buffer a second time to remove unbound antibody. The amount of bound antibody on the solid support is then determined according to well known methods. Those

skilled in the art will be able to determine optional assay conditions for each determination by employing routine experimentation.

One of the ways in which EF specific antibodies can be detectably labeled is by linking the antibody to an enzyme, such as for use in an enzyme immunoassay (EIA) (Voller, A.,"The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2: 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller, A., et al., 1978, J. Clin. Pathol. 31: 507-520; Butler, J. E., 1981, Meth. Enzymol. 73: 482-523). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or by visual means. Enzymes that can be used to detectable label the antibody include, but are not limited to, horseradish peroxidase and alkaline phosphatases. The detection can also be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme.

Detection of EF specific antibodies may also be accomplished using a variety of other methods. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect EF expression through the use of a radioimmunoassays (EIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

The antibody may also be labeled with a fluorescent compound.

Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Likewise, a bioluminescent compound may be used to label the EF antibody. The presence of a bioluminescence protein is determined by detecting the presence of luminescence.

Important bioluminescence compounds for purposes of labeling are luciferin, luciferase and aequorin.

In addition, reagents other than antibodies, such as, for example, polypeptides that bind specifically to EF can be used in assays to detect the level of EF expression.

5.1.4. KITS The present invention further provides for kits for carrying out the above-described assays. The assays described herein can be performed, for example, by utilizing pre-packaged diagnostic kits, comprising at least a EF antibody reagent (for detection of EF protein), which can be conveniently used, e. g., in clinical settings to diagnose endometriosis.

In a first series of nonlimiting embodiments, a kit according to the invention may comprise components which detect and/or measure EF antigen in the biological sample of a subject. For example, where EF is detected and/or measured by enzyme linked immunoabsorbent assay (ELISA), such components may comprise an antibody directed to epitopes of EF which can be used to detect and/or quantitate the level of EF expression in the biological sample. The antibody itself may be detectably labeled with a radioactive, flourescent, colorimetric or enzyme label. Alternatively, the kit may contain a labeled secondary antibody.

In a second series of nonlimiting embodiments, a kit according to the invention may comprise components which detect and/or measure EF mediated inhibition of PIF activity as measured by lymphocyte/platelet binding. Such components may comprise blood lymphocytes containing platelets, PIF activity (e. g., serum from a pregnant female), an anti-CD2 antibody and a control serum sample.

6. EXAMPLE: INCREASED LEVELS OF EF DETECTED IN THE SERUM OF ENDOMETRIOSIS PATIENTS The following subsection describes experimental data relating to the detection of increased levels of EF in the serum of endometriosis patients.

6.1. MATERIALS AND METHODS 6.1.1 CHEMICALS Guinea pig complement (GPC), Histopaque and Dulbecco's Phosphate Buffered Saline (PBS) were obtained from Sigma (St. Louis, MO).

6.1.2 PREPARATION OF LYMPHOCYTES Lymphocytes and platelets were obtained from healthy donors by a standard density gradient technique (Histopaque-1077; Sigma Chemical Co, St. Louis, MO, USA). Following centrifugation at 1500x for 20 minutes, the interface (lymphocytes) and two thirds of the supernatant (plasma enriched with platelets) were removed and combined in a 15 ml polypropylene tube. The cells were then washed with 0.01% bovine serum albumin in phosphate-buffered saline (0.01% BSA/PBS), pH 7.4 by centrifugation at 200x g for 10 minutes, and then resuspended in BSA/PBS to a density of ten million lymphocytes per ml.

6.1.3 LYMPHOCYTE/PLATELET BINDING ASSAYS Peritoneal fluid was collected during surgery, kept cold, and then centrifuged at 5000 rpm for 20 min to remove cell debris. The clear fluid was used for the assays. The serum samples were either used fresh or frozen at-20°C.

The lymphocyte/platelet binding assays were carried out in microcentrifuge tubes. 30 ßl of the cell suspension (lymphocytes plus platelets) was added to 20, ul of serum from first trimester pregnant women and the endometriosis sample to be tested. The sample was incubated at room temperature for 10 minutes, agitating every 2 minutes. 3 nl ofanti-CD2 monoclonal antibody (Ortho-Diagnostics, Raritan, N. J.) was then added and a second incubation was performed at room temperature for 10 minutes, gently agitating every 2 minutes for the first 6 minutes.

10, ul of the sample was withdrawn and placed on a slide with a cover slip. The number of lymphocytes with, or without, three or more bound platelets were counted.

A total of 200 cells in three different fields were counted. Samples were then recounted by placing another 10, ul of mixture on a slide and the average of the duplicate measurements was reported.

6.1.4. DATA ANALYSIS Data are expressed as percent platelet-bound lymphocytes per total bound and non-bound lymphocytes. Statistical analysis was carried out by using chi-

square analysis with Fischers Exact Test and ANOVA one way analysis of variance.

A difference of P <0.05 was considered significant.

6.1.5 GEL PERMEATION CHROMATOGRAPHY 0.5 ml sera sample is passed through a Shodex KW-803 column equilibrated with 0.1 % TFA in water (Buffer A). The column was run at 1 ml/min at a gradient of 0-100% buffer B (0.1 % TFA in 99.1% acetonitrile in 20 min. One ml fractions were collected starting at 3 min. The samples were dried, resuspended in 100 ul distilled water and assayed for EF activity.

6.1.6 REVERSE PHASE HPLC Samples were injected onto a C5 HPLC column equilibrated with 0.1 % TFA in water (Buffer A). The column was run at 1 ml/min at a gradient of 0-100% 0.1% TFA in 99.9% acetonitrile (Buffer B) was run in 20 min. One ml fractions were collected starting at 3 minutes. The samples were dried, resuspended in 100 ul distilled water and assayed for EF activity.

6.2. RESULTS In a total of 10 samples of patients with endometriosis, an inhibitory factor (s) was present that reduced the binding of lymphocytes to platelets compared to healthy sera controls. As indicated in Figures 1 and 2, increasing amounts (2,4,6 ul) of the endometriosis sera progressively inhibited the binding of lymphocytes to platelets. This effect was not noted when it was compared to buffer or sera used as controls. Assays using increasing doses of peritoneal fluid also caused a dose dependent reduction in binding of lymphocytes to platelets compared to controls (Figure 3).

Two samples derived from endometriosis patients were separated on a gel filtration HPLC, collecting individual fractions (Figure 4 and 5). The samples collected were lyophilized and resuspended in a small volume and tested in a lymphocyte/platelet binding assay (Figure 6). Inhibitory EF activity was found in the same fractions in both cases and suggest a size of approximately 2-7 kDa. Control

sera showed no inhibition.

Subsequently, reversed phase HPLC was run in both samples (Figure 7 and 8). Again, a very similar narrow EF inhibitory region was noted (Figure 9).

These results indicate that lymphocyte/platelet binding assays provide a method for identification of low molecular weight compounds expressed in endometriosis patients.

In addition, sera was progressively heated and tested for EF activity.

The results showed a temperature dependent decrease in the inhibitory activity. Anti- phospholipid antibodies also failed to inhibit the activity of EF.

The present invention is not to be limited in scope by the embodiments disclosed in the examples which are intended as an illustration of one aspect of the invention and any method which is functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Various publications are cited herein, the contents of which are incorporated by reference in their entirety.