The enzyme-linked immunosorbent assay for detection of the antispermatozoal antibodies

Vol. 38, No. 6, December 1982 Printed in U.SA.

FERTILITY AND STERILITY Copyright © 1982 The American Fertility Society

The enzyme-linked immunosorbent assay for detection of the antispermatozoal antibodies

Renato Zanchetta, M.D. *t Franco Busolo, Ph.D.:j: Ismaele Mastrogiacomo, M.D.* University of Padua, Medical School, Padua, Italy

An immune-enzymatic method was developed for the determination of antispermatozoal antibodies. Sera of 94 infertile patients were studied with the enzyme-linked immunosorbent assay (ELISA). Twelve patients were positive for antispermatozoal antibodies. Forty-six of these patients were studied also with the gelatin agglutination test (GAT). Eight sera were positive in the ELISA and nine in the GAT. With ELISA, immunoglobulin classes can be demonstrated; in fact, nine of our patients were positive for IgG and three for IgM. In all patients the IgA titer was less than 1:16. In addition, 61 seminal fluid specimens were studied by EUSA, and 7 were positive. The serum and seminal fluid of 12 patients were simultaneously studied. Seminal fluid was positive in only three patients, serum was positive in four, whereas serum and seminal fluid were both negative in five. This study illustrates that ELISA is apparently less sensitive than GAT; however, it is certainly more practical and an easier method for antibody research in sera and in seminal fluid. Fertil Steril 38:730, 1982

An immune-enzymatic method (enzyme-linked immunosorbent assay [ELISA]) was first described by Van Weemen and Shuurs, 1 and Engvall and Perlmann2 for the analytic study of the antigen-antibody reactions. This method uses an antigen or antibody, conjugated to an enzyme, and a solid phase as a support for one of the immunoreagents. The ELISA has found application in numerous fields of clinical medicine either for demonstration of antigens of infectious agents or of hormones, drugs, plasmatic and oncofetal antigens, antibodies (antibacteria, antivirus), and in aller-

Received March 9, 1982; revised and accepted July 12, 1982. *Istituto di Semeiotica Medica. tReprint requests: Renato Zanchetta, M.D., Istituto di Semeiotica Medica, Via Ospedale 105, Padua, Italy. tAssociate Professor, Institute of Microbiology.

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Zanchetta et al. Antisperm antibodies: ELISA

gology and clinical immunology (identification of immunoglobulins, antiallergens, autoantibodies, immunocomplexes). This method is also utilized for identification of antispermatozoal antibodies. These antibodies are present in sera and in seminal fluid. In the past few years many authors have affirmed the necessity of closely examining the seminal fluid; indeed, it is thought that seminal fluid may contain antispermatozoal antibodies able to cause sterility. 3 -5 Witkin et al. 6 applied this method in a study for demonstration of receptors of the Fe fragment of IgG on spermatozoa and their utilization for the detection of circulating immune complexes in human serum. In a further study using this method they 7 again showed the presence of IgA antibody to spermatozoa in human seminal fluid. Sucrosegradient velocity centrifugation demonstrated that the antisperm IgA in seminal fluid was US. Fertility and Sterility

A 1,3 1.2 1.1

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Figure 1 Background from a pool of negative sera (lgA, o; lgM, •; lgG, e).

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Figure 2 Background from a pool of negative sen nalfluids (lgA, o; IgG, e; lgM, •>·

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Figure 3 A serum with a higher titer oflgG (e) than lgM ~).The lgA (o) are present at a lower titer. A

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Figure 4 The lgM <•> and IgG {e) are present in a higher titer in the serum of another patient. The lgA (o) are present only at a low titer.

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Figure 15 A seminal fluid with a high titer of lgM <•> and with a low titer oflgG (e) and lgA

(o).

The aim of our study was to review a group of infertile patients and to compare the traditional antispermatozoal antibody research methods, particularly the gelatin agglutination test (GAT), with the results of ELISA, both in serum and in seminal fluid. MATERIALS AND METHODS

A total of94 men suffering from infertility were studied. Serum and seminal fluid were simultaneously studied in 12. Blood samples were drawn without anticoagulation and were clarified by centrifugation and stored at - 20° C. Sixty-one seminal fluid samples were studied as well. Vol. 38, No. 6, December 1982

18

1:4

8

16

32

84

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Figure 6 In this seminal fluid only the lgA (o) showed a titer > 1:64.

Sperm Agglutination Test. Sperm agglutinins in sera were determined by GAT' (minimal dilution 1:4). Sperm Immobilization Test. Sperm-immobilizing antibodies in sera were determined by the method of Isojima et al. 9 Sperm Cytotoxicity Test. The antibodies were determined by the method of Hamerlynck and Riimke. 10 EUSA. The pool of spermatozoa for the assay was obtained by fertile and mycoplasma-free donors. The search for mycoplasmas in seminal fluid was done by the Microbiology Institute of the University of Padua. Washed spermatozoa were adjusted with phosphate-buffered saline (PBS) to a concentration of Zanchetta et al. Antisperm antibodies: EUSA

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Table 1. Results of the GAT and the EUSA in the Sera of 46 Infertile Patients

Positive to high titer (> 1:64)

Positive to low titer (< 1:32)

No. of sera

GAT

ELISA

170 319 847 1189

+ +

+ + + +

95 124 174 210 228 412 850 1038 1050

+ + + + + + +

+ +

+ +

2.5 x 105 in 50-~J.l aliquots and fixed to wells of a microtiter plate (Cooke Engineering Co., Pool Bioanalysis ltaliana [PBI], Milan, Italy) with 0.25% glutaraldehyde. The fixed spermatozoa were washed three times with PBS containing 0.05% Tween 20 (Merck-n.822184, Darmstadt, Germany) and either used immediately or stored at -20° C. The enzyme-linked immunoassay procedure was the same described by Voller et al., 11 with some minor modifications. The sperm-coated wells were washed three times with PBS-Tween and a 50-j.Ll serum or seminal fluid specimen was added (dilution 1:4). The addition of Tween 20 eliminated nonspecific immunoglobulins binding to the wells. Samples were always assayed in duplicate, and the positive sera were assayed in serial dilution in PBS. After a 60-minute incubation at 37° C, the wells were washed three times with PBS-Tween, and 50 ,...1 of a 1:100 dilution in PBS of alkaline phosphatase-conjugated goat anti-human immunoglobulins of the classes lgG, lgA, and lgM, and separately with anti-human lgG, IgA, and lgM, was added. Following a 60-minute incubation at 37° C the wells were again washed three times in PBS-Tween, and 250 ,...I of 1 mg/ml solution of p-nitrophenyl phosphate in 0.05 M sodium carbonate buffer (pH 9.8) was added. After a 60-minute incubation at 37° C, 50 ,...I of 3 N NaOH was added to each well to stop the reaction. The absorbance was determined at 400 nm in a Perkin-Elmer 124 double-ray spectrophotometer (Perkin-Elmer, Norwalk, CT) using microcuvettes. A blank consisting of p-nitrophenyl phosphate and 3 N NaOH was assayed in wells parallel to the experimental samples. The titer is expressed by the highest dilution that gives absorbance values greater than those 732

Zanchetta et al. Antisperm antibodies: EUSA

determined for negative antibody samples and background (Figs. 1 and 2). RESULTS

Of the 94 sera of infertile patients studied with ELISA, 12 (12.7%) were positive. Eight (8.5%) sera were positive at low titer (from 1:4 to 1:16), while another four (4.2%) were positive with titers varying between 1:32 to more than 1:64. Table 1 shows that 46 (48.9%) patients were studied with two methods: GAT for agglutinating antibodies and ELISA. Eight (1 7.3%) sera were positive for ELISA and nine (19.5%) for GAT. Three positive sera in the GAT were positive also in the ELISA. Only one positive sample in the GAT was weakly(< 1:4) positive in the ELISA, while only one positive (1:64) serum in the ELISA was negative in the GAT, but positive (1:4) in the sperm cytotoxicity test. Five positive (1:4) patients in the GAT were negative in the ELISA. Three patients, on the contrary, weakly ( < 1:8) positive in the ELISA, were negative in the GAT, but weakly (1:4) positive in the sperm immobilization and sperm cytotoxicity tests. The 12 positive sera in the ELISA were studied for the demonstration of the different classes of immunoglobulins with goat hyperimmune antihuman lgA, IgG, and lgM serum. Nine (75%) patients showed a titer 1:64 of IgG, three (25%) showed a titer > 1:64 of IgM, and in all patients the lgA titer was < 1:16 (Figs. 3 and 4). In addition, 61 seminal fluids were studied with ELISA: 7 (11.4%) were positive. Five patients showed low (1:4) titers, while one patient showed a higher (1:64) titer for lgA and another patient showed a titer for lgM of 1:32 (Figs. 5 and 6). Finally, 12 patients were studied simultaneously for serum and seminal fluid for detection of antispermatozoal antibodies: 3 were positive only for seminal fluid, 4 were positive only for serum, and 5 were negative for both serum and seminal fluid. DISCUSSION

The agglutination method has been used for many years in various laboratories for research in antispermatozoal antibodies in serum and seminal fluid, and it has been used satisfactorily in our laboratory. However, it is time-consuming and difficult to find an antigen that is always valid for the test, Fertility and Sterility

since the spermatozoa must be fresh, motile, and numerous. In our study, the comparative evaluation between ELISA and GAT has shown that the greatest advantage of ELISA is that is is easy and practical both for serum and seminal fluid, although apparently less sensitive. In fact, it uses a smaller pool of spermatozoa, which does not necessarily have to be motile for every well of amicrotiter plate. This solid phase can be preserved for long periods in a freezer without damage to antigenic properties. Moreover, with the use of the microtiter plates it is possible to analyze simultaneously many samples of different dilutions. With regard to the minor sensitivity of the ELISA method, one may consider it to be only relative, since the nonspecific microagglutinations that often interfere in the GAT are not present in the ELISA method. With the GAT it is not always easy to distinguish the microagglutinations because of the presence of round cells, debris,5 bacteria, or mycoplasmas. Unexplained autoagglutination in ejaculate could be caused by an infection with mycoplasmas or ureaplasmas. 12 Furthermore, with ELISA it is possible to make a more objective determination of antibody titers. The positive samples can be immediately seen by naked-eye evaluation, and the antibody titer can be determined by use of the spectrophotometer for a curve of extinction of the absorbance. Moreover, by using class-specific antisera, ELISA permits the determination of immunoglobulin classes that could indicate the onset of antibody production. In our study two patients had serum titers of IgM that were higher than those of IgG or IgA. This presence of IgM could be an important marker in dating the onset of antibody production and the activation of complement. Studying seminal fluid by ELISA, we found seven positive patients, one of which had a high titer of IgA (> 1:64), and our results are in agreement with those of Witkin et al. 7 The precise location of production of this secretory IgA is still a matter of speculation, but it could be the prostate gland, because the presence of a subclinical pathologic condition could lead to sperm extravasation. These seminal fluid antibodies could interfere with conception both by agglutination and/or immobilization of spermatozoa in the ejaculate or by interference with sperm transport through cervical mucus. 13 Vol. 38, No. 6, December 1982

Although IgM is not present in normal seminal fluid, 14· 15 we have found, in accordance with Kula et al., 16 one patient with IgM (1:32) in seminal fluid. Some patients, because of repeated inflammations, could have alterations of testicular vascular structures that could explain the presence of IgM in seminal fluid. IgM in seminal fluid may damage spermatozoa by the activation of complement. In conclusion, we think that ELISA may be useful in the clinical evaluation and in follow-up to therapy for the measurement of antispermatozoal antibodies in sera and semen. This method is rapid, practical, inexpensive, and reproducible and yields nonsubjective determinations of antibody levels. It can be associated with the more traditional methods of research in antispermatozoal antibodies.

REFERENCES 1. Van Weemen BK, Shuurs AMWM: Immunoassay using antigen-enzyme conjugates. FEBS Lett 15:232, 1971 2. Engvall E, Perlmann P: Enzyme linked immunosorbent assay (ELISA): quantitative assay of immunoglobulin G. Immunochemistry 8:871, 1971 3. Friberg J: Immunological studies on human spermagglutinating seminal fluid. Acta Obstet Gynecol Scand (Suppl) 36:65, 1974 4. Husted S, lfjort B: Sperm antibodies in serum and seminal plasma. Int J Fertil20:97, 1975 5. Quinlivan WLG, Sullivan H: Antispermatozoal effects of human seminal plasma-an immunologic phenomenon. Fertil Steril 27:1194, 1976 6. Witkin SS, Shahani SK, Gupta S, Good RA, Noorbibi KD: Demonstration of lgG Fe receptors on spermatozoa and their utilization for the detection of circulating immune complexes in human serum. Clin Exp Immunol 41:441, 1980 7. Witkin SS, Zelikovsky G, Good RA, Noorbibi KD: Demonstration of 11S lgA antibody to spermatozoa in human seminal fluid. Clin Exp Immunol 44:368, 1981 8. Kibrick S, Belding DL, Merrill B: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, 1952 9. Isojima S, Li TS, Ashitaka Y: Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility. AmJ Obstet Gynecol101:607, 1968 10. Hamerlynck J, Riimke P: A test for the detection of cytotoxic antibodies to spermatozoa in men. J Reprod Fertil 17:191, 1968 11. Voller A, Bidwell D, Bartless A: Microplate enzyme immunoassay for the immunodiagnosis of virus infections. In Manual of Clinical Immunology, Edited by N Rose, W Friedman. Washington, D.C., American Society for Microbiology, 1976, p 506 12. Friberg J: Mycoplasmas and ureaplasmas in infertility and abortion. Fertil Steril 33:351, 1980 13. Kremer J, Jager S: The sperm-cervical mucus contact test: a preliminary report. Fertil Steril 27:335, 1976

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14. Coombs RRA, Riimke Ph, Edwards RG: Immunoglobulin classes reactive with spermatozoa in the serum and seminal plasma of vasectomised and infertile men. In the Second International Symposium on Immunology of Reproduction, Edited by K Bratanov, RG Edwards, VH Vulchanov, V Dikov, B Somlev. Sofia, Bulgarian Academy of Science Press, 1973, p 354

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15. Hjort T: Immunological capacity of the male genital tract. In Immunological Influences of Human Fertility, Edited by B ~oettcher. Sydney, Academic Press, 1977, p 115 16. Kula K, Owczarczyk I, Reterski Z: Serum immunoglobulins lgG and lgM in the seminal plasma of men with normospermia, oligospermia and azoospermia. Arch Androl 3:37, 1979

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