- Email: [email protected]
Detection of anti-spermatozoal antibodies by a 125I-protein-A radioimmunobinding assay
Journal of Reproductive Immunology, 8 (1985) 313-319
313
Elsevier JRI 00394
Detection of anti-spermatozoal antibodies by a 125I-protein-A radioimmunobinding assay A.B. C z u p p o n Institute for Monoclonal Antibodies, Department of Clinical Immunology, Feodor Lynnen Strasse 5, D-3000 Hannover, F.R. G.
A newly developed solid-phase radioimmunobinding assay (RIBA) using detergent-solubilized sperm antigen has been used to evaluate anti-sperm antibodies. Results showed that none of the fertile females and males were positive in the RIBA, whereas approximately 6% of the females and 3% of the males with unexplained infertility were positive. These results are similar to those obtained using a chemically synthesized spermatozoal decapeptide antigen. The RIBA is simple to perform, requires no vital or intact spermatozoa, and large numbers of sera (up to 400) can be processed in one day with a total incubation time of 90 min. Key words: anti-sperm antibodies, radioimmunobinding assay, solubilized spermatozoal membranes
Introduction
Ever since Landsteiner (1899) discovered the antigenic properties of human spermatozoa there have been numerous tests devised for the diagnosis of immunological infertility (Husted and Hjort, 1974; Noyes et al., 1974; Mathur et al., 1981). Although the etiology of such iso- and autoantibodies is still unknown it is established that their presence could be responsible for infertility (McLaren, 1964; Mettler et al., 1983). The main difficulties for an adequate diagnostic test lie in the preparation of the proper antigen(s) to be used, as well as in the detection methods for the reacting antibodies. Most of the tests that are in use require either motile spermatozoa or fixed intact cells and thus are dependent upon a regular supply of cells. In addition, they rely on a subjective interpretation, which makes an objective comparison between various investigators difficult. Furthermore, some of these tests are known to be influenced by non-specific factors (Boettcher, 1974). Theoretically, the intact spermatozoal membrane might not be the best choice of target antigen in testing for anti-spermatozoal antibodies since many of the relevant antigens could either be masked on the intact surface (Czuppon, 1983) or not expressed at all (Mettler et al., 1983). In the present study, spermatozoal membranes were first solubilized by the 'zwitter-ionic' detergent 3-(-(cholamidopropyl)-dimethylammonio}-propanesulfonate (CHAPS). The solubilized membrane mixture was used as antigen following immobilization on polystyrene beads. Human serum 0165-0378/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
314 antibodies were then detected with the aid of 125I-protein-A. The anti-spermatozoal antibody concentrations were directly calculated from the specific activity of the 125I-protein-A and its binding capacity. Antigen-coated beads can be prepared in advance and stored for up to 1 year when kept at 4°C. This eliminates the need for fresh antigen preparations.
Methods
Solubilization of the spermatozoal antigens The spermatozoal membrane was solubilized by the procedure described by Czuppon and Mettler (1982a), except for the presence of 10% (w/v) CHAPS. The detergent was removed by dialysis against 0.05 M barbital buffer, pH 9.0, for 72 h. The dialysate was then lyophilized and dissolved in the above buffer. After filtration through a Millipore filter (0.45 #) the protein concentration was adjusted to 9 mg/ml. This solution of spermatozoal membrane antigens was used to coat the polystyrene beads for the RIBA. Aliquots of cell suspensions before and after detergent treatment were also taken for transmission electron-microscopy (TEM).
Synthetic spermatozoal decapeptide antigen The chemical synthesis of one of the antigen (hapten) analogues in the solubilized spermatozoal membrane mixture has been described earlier (Czuppon and Mettler, 1982). A 1 m g / m l solution of the synthetic peptide in 0.05 M barbital buffer was used to coat the polystyrene beads.
Preparation of the antigen-coated beads The polystyrene beads (3 mm diameter) were incubated in the antigen solution (60 beads/ml) with gentle shaking for 120 min. This was followed by overnight incubation at 4°C. Following incubation the beads were washed with ice-cold 0.05 M barbital buffer and dried at 37°C. The coated beads were then stored at 4°C until use.
1251-protein-A The 1251labelled protein-A was obtained from New England Nuclear, Dreieich, F.R.G. The specific activity was 7-8 n C i / n g and the IgG binding capacity 10 n g / n g protein-A.
Radioimmunobinding assay (RIBA) Basically, the RIBA was the same as that described previously for whole spermatozoa (Czuppon and Mettler, 1982) and for the synthetic decapeptide antigen (Czuppon and Mettler, 1982). Frozen samples, were tested. The sera were diluted 1 : 100 with 0.03 M phosphate buffer, pH 7.2, and incubated with the beads in RIA tubes for 30 min at 37°C. This was followed by incubation for 30 min at 25°C. After incubation the solutions were pipetted off and the beads washed twice with 1 ml of 0.05 M barbital buffer. 200 /~1 of the 125I-protein-A solution (60,000 cpm) was then pipetted to each tube in 0.05 M barbital buffer, pH 9.0, containing 0.01% BSA. The tubes were then incubated again for 30 min at 37°C and the washing procedure repeated. Each tube was then counted for 1 min in a y-counter. All assays were carried out in triplicate. With each
315 test a positive control serum (rabbit anti-human spermatozoal antiserum; 1 : 100, 1 : 1,000, 1 : 2,000, 1 : 4,000 and 1 : 8,000 dilutions) was included to maintain quality control. The b o u n d anti-spermatozoal I g G concentrations were calculated according to the equation ng I g G = (average cpm x bc) x (1,268 x sa)-l; where bc = binding capacity of protein-A in ng; sa = specific activity of protein-A in n C i / n g . A n I g G level of 7.8 ng or greater was considered positive for female sera, 8.0 ng for the male sera, according to the upper levels in the negative control groups (see below). The cut-off levels for the synthetic decapeptide antigen were set at 300 and 200 ng respectively.
Results
Solubilized spermatozoal antigens The T E M of the spermatozoa before and after detergent treatment indicated that most of the acrosomal end equatorial membranes were solubilized (Fig. 1). Sera of females There were no positive reactions a m o n g the sera of fertile females and several other female categories also revealed purely negative results (post-partum females, post-menopausal females, primary amenorrhoea, a u t o i m m u n e ovarian failure and virgins). In the category of unexplained infertility, however, 4 of 69 sera showed an elevated anti-spermatozoal I g G level (Table 1), and 2 of 11 sera in the category of recurrent abortions were also found to be positive. The 4 patients that were positive in the unexplained infertile category included two primary sterility
TABLE 1 Results with the female sera in RIBA using solubilized spermatozoal antigen Category
No. positive reactions a
No. negative reactions
1 Fertile females 3 Unexplained infertility 5 Pregnant women 6 Post-partum women 8 Females with recurrent abortion 9 Virgins 10 Post-menopausal women 11 Primary amenorrhoea 13 Auto-immune ovarian failure
0 (0) 4
23 (10) 65
1 (I) 0 (0) 2 (0)
30 (2) 14 (5) 9 (1)
0 0
9 7
0 (0) 0
2 1
a The values in brackets represent results with the synthetic antigen in the RIBA.
316
Fig. 1. Electron-micrograph of human solubilization in 10% CHAPS. Note membranes following the solubilization
spermatozoa (X 25,000). Right, before solubilization; the complete disintegration of the acrosomal and procedure.
left, after equatorial
cases of 6 yr duration, one primary sterility persisting for 8 yr and a secondary infertility lasting 2 yr. The female patient with secondary sterility showed the highest level of anti-spermatozoa1 IgG (11.8 ng) compared with all the other categories and also had a husband with an elevated anti-spermatozoa1 IgG level (7.9 ng), which is about 2.5 times the average of the fertile males.
No positives were found amongst the sera from fertile men or men Seru of males with aspermatogenesis or congenital absence of the vas. One out of 13 of the vasectomized men and 2 out of 73 with unexplained infertility were positive (Table 2).
317 TABLE 2 Results with the male sera in RIBA using solubilized spermatozoal antigen Category
Number positive reactions a
Number negative reactions
2 Fertile males 4 Unexplained infertility 14 Vasectomizedmen 15 Congenital absence of vas 16 Aspermatogenesis
0 (0) 2 (2)
18 (10) 73 (25)
1 (0) 0 (0) 0 (0)
13 (13) 3 (3) 4 (4)
a The values in brackets represent results with the synthetic antigen in the RIBA.
Duplicates
An intra-pair comparison of the ngIgG levels of the duplicates by the Spearman rank correlation coefficient test indicated no significant deviations of the values in the duplicates (r S = 0.55, n = 24, P < 0.01).
Discussion It can be seen from the electron micrograph that the acrosomal and equatorial membranes of spermatozoa are completely solubilized by CHAPS, while the rest of the spermatozoal head remains intact. In addition, cross-sections of the spermatozoal tail showed that these surface membranes are also dissolved by the detergent. It can therefore be assumed that the coated beads represent an artificial spermatozoal 'surface membrane', the main difference being the arrangement of the membrane components. Furthermore, since the binding of the various membrane components to the beads is a hydrophilic interaction at alkaline pH, the hydrophobic molecules of solubilized spermatozoal membranes should bind preferentially to the beads. It was shown previously that the iso- and auto-antibodies reactive with spermatozoa that are present in the sera of some patients with unexplained infertility do react with protein-A (Skrabei et al., 1980). The advantage of using radio-labelled protein-A as a tracer becomes obvious when one considers the specific activity of the labelled compound (i.e., nCi/ng). There are 14 lysine residues in the molecule and all the c-amino groups are available to radioactive iodine (125I) introduced by the Bolton-Hunter reagent. Together with the N-terminal amino acid residue, this makes it possible to introduce 15 molecules of 125I per molecule of protein-A, thus giving a very high specific activity. The RIBA is simple to perform and an adequate quality control can be obtained by including serial dilutions of a known control serum with each assay. The frequency of positive sera in both the female and male categories is as expected, although the 3% in the unexplained male infertility group seems somewhat low. One pregnant female was found to be positive out of 30 sera in that category, but the same female was also found to be positive by most other laboratories. Fertiles, post-partum females and virgins are generally taken as better negative controls.
318 It is interesting to note the reactions with the synthetic decapeptide. While no positive sera were found in the control groups, approximately 8% of the unexplained infertility categories for both females and males were positive. This is a higher frequency than that obtained with the spermatozoal membrane autolysate as antigen. Furthermore, sera that were positive with one antigen were negative with the other. There are several possible explanations for this discrepancy. The most likely is that the avidity of the naturally occurring antibodies is higher for the synthetic peptide antigen than for the autolysate, which is a mixture of various antigens, and also the native decapeptide is less than 10% of the total peptide/protein content in the autolysate (Czuppon and Mettler, 1982b). A more important point seems to be the lack of reaction of the synthetic decapeptide with sera that are positive with the spermatozoal membrane autolysate. It is most likely that the naturally occurring anti-spermatozoal antibodies in the sera of patients are induced by different antigens. This has already been postulated in previous work (Czuppon and Mettler, 1982) where anti-spermatozoal antibodies in some patients could be absorbed with the synthetic decapeptide antigen, while in others a lack of reaction was noted. A multifactorial cause of immunologically-induced infertility, i.e., induction of antibodies by different antigens, becomes more evident when other antigens are used in the RIBA. For example, when zona-pellucida antigens are substituted in the assay the frequency of positive sera in the unexplained infertility categories increases to around 13-14% and include sera which fail to react with spermatozoal antigens (Czuppon, 1984). It seems very likely that no test can detect all forms of immunological infertility when only one type of target antigen is used. This implies that the true frequency of immunological infertility amongst unexplained infertility cases is higher than that previously assumed. Finally, the results of the duplicate samples should be mentioned. Plotting the values (ngIgG) of the 24 pairs against each other, a significant intra-pair correlation was obtained, indicating the reliability of the RIBA results. Indeed, it appears that the newly introduced RIBA could provide a useful addition to laboratory tests for immunological infertility in both males and females.
References Boettcher, B. and Kay, D.J. (1974) Agglutination of spermatozoa by human sera with added steroids. Andrologia 5, 265-273. Czuppon, A.B. (1984) Biochemical characterization of a human spermatozoal sialoglycoprotein with respect to antigenicity masking by its sialic acid moieties. BiochemistryInt. 8, 9-18. Czuppon, A.B. and Mettler, L. (1982a) Acrosin-dependent solubilization of antigenic peptides from human spermatozoa. 1, 303-306. Czuppon, A.B. and Mettler, L. (1982b) Chemical synthesis of a decapeptide eliciting characteristics of a human spermatozoal antigen. Hoppe-Seyler'sZ. Physiol. Chem. 363, 1465-1471. Czuppon, A.B. and Mettler, L. (1983) Estimation of anti-spermatozoa antibody concentrations by a 125I-protein-A binding assay in sera of infertile patients. Clin. Chem. Clin. Biochem. 21,357-362. Husted, S. and Hjort, T. (1974) Microtechnique for detection of sperm immobilizationand cytotoxicity. WHO-Workshop on techniques for detection of iso- and autoantibodies to human spermatozoa. Aarhus, Denmark, July 15-19. Scriptor, Denmark.
319 Landsteiner, K. (1899) Zur Kenntnis der spezifisch auf Blutk6rperchen wirkenden Seren. Ztr. B1. Bakt. orig. A. 25, 546-549. Matthur, S., Williamson, H.O., Derick. F.C., Madystha, R.P., Melcho, J.T., Holtz, G.L., Baker, E.R., Smith, C.L. and Fudenberg, H.H. (1981) A new microassay for spermacytotoxic antibody: comparison with passive haemagglutination assay for antisperm antibodies in couples with unexplained infertility. J. Immunol. 126, 905-909. Mettler, L., Czuppon, A.B., Buchheim, H., Baukloh. V., Chyczy, M., Etschenberg, J. and Holstein, A.F. (1983) Induction of high titer mouse-antihuman spermatozoal antibodies by liposome incorporation of spermatozoal membrane antigens. Am. J. Reprod. Immunol. 4, 127-132. Mettler, L., Czuppon, A.B. and Tinneberg, H.-R. (1983) Immunization with spermatozoal peptide antigens resulting in immuno-suppression of fertility rates in female rats. Andrologia 15, 670-675. McLaren, A. (1964) Immunological control of fertility in female mice. Nature (London) 201, 582-585. Noyes, R.W., Odom, C.B. and Camyre, M.R. (1983) Radioisotope assay of sperm-binding globulin. Contraception 7, 57-59. Skrabei, H, Czuppon, A.B. and Mettler, L. (1980) Reaction of human spermatozoal haptens with sperm antibody containing sera of sterile males and females (protein-A tracer RIA) Arch. Androl. 5, 13-15.
313
Elsevier JRI 00394
Detection of anti-spermatozoal antibodies by a 125I-protein-A radioimmunobinding assay A.B. C z u p p o n Institute for Monoclonal Antibodies, Department of Clinical Immunology, Feodor Lynnen Strasse 5, D-3000 Hannover, F.R. G.
A newly developed solid-phase radioimmunobinding assay (RIBA) using detergent-solubilized sperm antigen has been used to evaluate anti-sperm antibodies. Results showed that none of the fertile females and males were positive in the RIBA, whereas approximately 6% of the females and 3% of the males with unexplained infertility were positive. These results are similar to those obtained using a chemically synthesized spermatozoal decapeptide antigen. The RIBA is simple to perform, requires no vital or intact spermatozoa, and large numbers of sera (up to 400) can be processed in one day with a total incubation time of 90 min. Key words: anti-sperm antibodies, radioimmunobinding assay, solubilized spermatozoal membranes
Introduction
Ever since Landsteiner (1899) discovered the antigenic properties of human spermatozoa there have been numerous tests devised for the diagnosis of immunological infertility (Husted and Hjort, 1974; Noyes et al., 1974; Mathur et al., 1981). Although the etiology of such iso- and autoantibodies is still unknown it is established that their presence could be responsible for infertility (McLaren, 1964; Mettler et al., 1983). The main difficulties for an adequate diagnostic test lie in the preparation of the proper antigen(s) to be used, as well as in the detection methods for the reacting antibodies. Most of the tests that are in use require either motile spermatozoa or fixed intact cells and thus are dependent upon a regular supply of cells. In addition, they rely on a subjective interpretation, which makes an objective comparison between various investigators difficult. Furthermore, some of these tests are known to be influenced by non-specific factors (Boettcher, 1974). Theoretically, the intact spermatozoal membrane might not be the best choice of target antigen in testing for anti-spermatozoal antibodies since many of the relevant antigens could either be masked on the intact surface (Czuppon, 1983) or not expressed at all (Mettler et al., 1983). In the present study, spermatozoal membranes were first solubilized by the 'zwitter-ionic' detergent 3-(-(cholamidopropyl)-dimethylammonio}-propanesulfonate (CHAPS). The solubilized membrane mixture was used as antigen following immobilization on polystyrene beads. Human serum 0165-0378/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
314 antibodies were then detected with the aid of 125I-protein-A. The anti-spermatozoal antibody concentrations were directly calculated from the specific activity of the 125I-protein-A and its binding capacity. Antigen-coated beads can be prepared in advance and stored for up to 1 year when kept at 4°C. This eliminates the need for fresh antigen preparations.
Methods
Solubilization of the spermatozoal antigens The spermatozoal membrane was solubilized by the procedure described by Czuppon and Mettler (1982a), except for the presence of 10% (w/v) CHAPS. The detergent was removed by dialysis against 0.05 M barbital buffer, pH 9.0, for 72 h. The dialysate was then lyophilized and dissolved in the above buffer. After filtration through a Millipore filter (0.45 #) the protein concentration was adjusted to 9 mg/ml. This solution of spermatozoal membrane antigens was used to coat the polystyrene beads for the RIBA. Aliquots of cell suspensions before and after detergent treatment were also taken for transmission electron-microscopy (TEM).
Synthetic spermatozoal decapeptide antigen The chemical synthesis of one of the antigen (hapten) analogues in the solubilized spermatozoal membrane mixture has been described earlier (Czuppon and Mettler, 1982). A 1 m g / m l solution of the synthetic peptide in 0.05 M barbital buffer was used to coat the polystyrene beads.
Preparation of the antigen-coated beads The polystyrene beads (3 mm diameter) were incubated in the antigen solution (60 beads/ml) with gentle shaking for 120 min. This was followed by overnight incubation at 4°C. Following incubation the beads were washed with ice-cold 0.05 M barbital buffer and dried at 37°C. The coated beads were then stored at 4°C until use.
1251-protein-A The 1251labelled protein-A was obtained from New England Nuclear, Dreieich, F.R.G. The specific activity was 7-8 n C i / n g and the IgG binding capacity 10 n g / n g protein-A.
Radioimmunobinding assay (RIBA) Basically, the RIBA was the same as that described previously for whole spermatozoa (Czuppon and Mettler, 1982) and for the synthetic decapeptide antigen (Czuppon and Mettler, 1982). Frozen samples, were tested. The sera were diluted 1 : 100 with 0.03 M phosphate buffer, pH 7.2, and incubated with the beads in RIA tubes for 30 min at 37°C. This was followed by incubation for 30 min at 25°C. After incubation the solutions were pipetted off and the beads washed twice with 1 ml of 0.05 M barbital buffer. 200 /~1 of the 125I-protein-A solution (60,000 cpm) was then pipetted to each tube in 0.05 M barbital buffer, pH 9.0, containing 0.01% BSA. The tubes were then incubated again for 30 min at 37°C and the washing procedure repeated. Each tube was then counted for 1 min in a y-counter. All assays were carried out in triplicate. With each
315 test a positive control serum (rabbit anti-human spermatozoal antiserum; 1 : 100, 1 : 1,000, 1 : 2,000, 1 : 4,000 and 1 : 8,000 dilutions) was included to maintain quality control. The b o u n d anti-spermatozoal I g G concentrations were calculated according to the equation ng I g G = (average cpm x bc) x (1,268 x sa)-l; where bc = binding capacity of protein-A in ng; sa = specific activity of protein-A in n C i / n g . A n I g G level of 7.8 ng or greater was considered positive for female sera, 8.0 ng for the male sera, according to the upper levels in the negative control groups (see below). The cut-off levels for the synthetic decapeptide antigen were set at 300 and 200 ng respectively.
Results
Solubilized spermatozoal antigens The T E M of the spermatozoa before and after detergent treatment indicated that most of the acrosomal end equatorial membranes were solubilized (Fig. 1). Sera of females There were no positive reactions a m o n g the sera of fertile females and several other female categories also revealed purely negative results (post-partum females, post-menopausal females, primary amenorrhoea, a u t o i m m u n e ovarian failure and virgins). In the category of unexplained infertility, however, 4 of 69 sera showed an elevated anti-spermatozoal I g G level (Table 1), and 2 of 11 sera in the category of recurrent abortions were also found to be positive. The 4 patients that were positive in the unexplained infertile category included two primary sterility
TABLE 1 Results with the female sera in RIBA using solubilized spermatozoal antigen Category
No. positive reactions a
No. negative reactions
1 Fertile females 3 Unexplained infertility 5 Pregnant women 6 Post-partum women 8 Females with recurrent abortion 9 Virgins 10 Post-menopausal women 11 Primary amenorrhoea 13 Auto-immune ovarian failure
0 (0) 4
23 (10) 65
1 (I) 0 (0) 2 (0)
30 (2) 14 (5) 9 (1)
0 0
9 7
0 (0) 0
2 1
a The values in brackets represent results with the synthetic antigen in the RIBA.
316
Fig. 1. Electron-micrograph of human solubilization in 10% CHAPS. Note membranes following the solubilization
spermatozoa (X 25,000). Right, before solubilization; the complete disintegration of the acrosomal and procedure.
left, after equatorial
cases of 6 yr duration, one primary sterility persisting for 8 yr and a secondary infertility lasting 2 yr. The female patient with secondary sterility showed the highest level of anti-spermatozoa1 IgG (11.8 ng) compared with all the other categories and also had a husband with an elevated anti-spermatozoa1 IgG level (7.9 ng), which is about 2.5 times the average of the fertile males.
No positives were found amongst the sera from fertile men or men Seru of males with aspermatogenesis or congenital absence of the vas. One out of 13 of the vasectomized men and 2 out of 73 with unexplained infertility were positive (Table 2).
317 TABLE 2 Results with the male sera in RIBA using solubilized spermatozoal antigen Category
Number positive reactions a
Number negative reactions
2 Fertile males 4 Unexplained infertility 14 Vasectomizedmen 15 Congenital absence of vas 16 Aspermatogenesis
0 (0) 2 (2)
18 (10) 73 (25)
1 (0) 0 (0) 0 (0)
13 (13) 3 (3) 4 (4)
a The values in brackets represent results with the synthetic antigen in the RIBA.
Duplicates
An intra-pair comparison of the ngIgG levels of the duplicates by the Spearman rank correlation coefficient test indicated no significant deviations of the values in the duplicates (r S = 0.55, n = 24, P < 0.01).
Discussion It can be seen from the electron micrograph that the acrosomal and equatorial membranes of spermatozoa are completely solubilized by CHAPS, while the rest of the spermatozoal head remains intact. In addition, cross-sections of the spermatozoal tail showed that these surface membranes are also dissolved by the detergent. It can therefore be assumed that the coated beads represent an artificial spermatozoal 'surface membrane', the main difference being the arrangement of the membrane components. Furthermore, since the binding of the various membrane components to the beads is a hydrophilic interaction at alkaline pH, the hydrophobic molecules of solubilized spermatozoal membranes should bind preferentially to the beads. It was shown previously that the iso- and auto-antibodies reactive with spermatozoa that are present in the sera of some patients with unexplained infertility do react with protein-A (Skrabei et al., 1980). The advantage of using radio-labelled protein-A as a tracer becomes obvious when one considers the specific activity of the labelled compound (i.e., nCi/ng). There are 14 lysine residues in the molecule and all the c-amino groups are available to radioactive iodine (125I) introduced by the Bolton-Hunter reagent. Together with the N-terminal amino acid residue, this makes it possible to introduce 15 molecules of 125I per molecule of protein-A, thus giving a very high specific activity. The RIBA is simple to perform and an adequate quality control can be obtained by including serial dilutions of a known control serum with each assay. The frequency of positive sera in both the female and male categories is as expected, although the 3% in the unexplained male infertility group seems somewhat low. One pregnant female was found to be positive out of 30 sera in that category, but the same female was also found to be positive by most other laboratories. Fertiles, post-partum females and virgins are generally taken as better negative controls.
318 It is interesting to note the reactions with the synthetic decapeptide. While no positive sera were found in the control groups, approximately 8% of the unexplained infertility categories for both females and males were positive. This is a higher frequency than that obtained with the spermatozoal membrane autolysate as antigen. Furthermore, sera that were positive with one antigen were negative with the other. There are several possible explanations for this discrepancy. The most likely is that the avidity of the naturally occurring antibodies is higher for the synthetic peptide antigen than for the autolysate, which is a mixture of various antigens, and also the native decapeptide is less than 10% of the total peptide/protein content in the autolysate (Czuppon and Mettler, 1982b). A more important point seems to be the lack of reaction of the synthetic decapeptide with sera that are positive with the spermatozoal membrane autolysate. It is most likely that the naturally occurring anti-spermatozoal antibodies in the sera of patients are induced by different antigens. This has already been postulated in previous work (Czuppon and Mettler, 1982) where anti-spermatozoal antibodies in some patients could be absorbed with the synthetic decapeptide antigen, while in others a lack of reaction was noted. A multifactorial cause of immunologically-induced infertility, i.e., induction of antibodies by different antigens, becomes more evident when other antigens are used in the RIBA. For example, when zona-pellucida antigens are substituted in the assay the frequency of positive sera in the unexplained infertility categories increases to around 13-14% and include sera which fail to react with spermatozoal antigens (Czuppon, 1984). It seems very likely that no test can detect all forms of immunological infertility when only one type of target antigen is used. This implies that the true frequency of immunological infertility amongst unexplained infertility cases is higher than that previously assumed. Finally, the results of the duplicate samples should be mentioned. Plotting the values (ngIgG) of the 24 pairs against each other, a significant intra-pair correlation was obtained, indicating the reliability of the RIBA results. Indeed, it appears that the newly introduced RIBA could provide a useful addition to laboratory tests for immunological infertility in both males and females.
References Boettcher, B. and Kay, D.J. (1974) Agglutination of spermatozoa by human sera with added steroids. Andrologia 5, 265-273. Czuppon, A.B. (1984) Biochemical characterization of a human spermatozoal sialoglycoprotein with respect to antigenicity masking by its sialic acid moieties. BiochemistryInt. 8, 9-18. Czuppon, A.B. and Mettler, L. (1982a) Acrosin-dependent solubilization of antigenic peptides from human spermatozoa. 1, 303-306. Czuppon, A.B. and Mettler, L. (1982b) Chemical synthesis of a decapeptide eliciting characteristics of a human spermatozoal antigen. Hoppe-Seyler'sZ. Physiol. Chem. 363, 1465-1471. Czuppon, A.B. and Mettler, L. (1983) Estimation of anti-spermatozoa antibody concentrations by a 125I-protein-A binding assay in sera of infertile patients. Clin. Chem. Clin. Biochem. 21,357-362. Husted, S. and Hjort, T. (1974) Microtechnique for detection of sperm immobilizationand cytotoxicity. WHO-Workshop on techniques for detection of iso- and autoantibodies to human spermatozoa. Aarhus, Denmark, July 15-19. Scriptor, Denmark.
319 Landsteiner, K. (1899) Zur Kenntnis der spezifisch auf Blutk6rperchen wirkenden Seren. Ztr. B1. Bakt. orig. A. 25, 546-549. Matthur, S., Williamson, H.O., Derick. F.C., Madystha, R.P., Melcho, J.T., Holtz, G.L., Baker, E.R., Smith, C.L. and Fudenberg, H.H. (1981) A new microassay for spermacytotoxic antibody: comparison with passive haemagglutination assay for antisperm antibodies in couples with unexplained infertility. J. Immunol. 126, 905-909. Mettler, L., Czuppon, A.B., Buchheim, H., Baukloh. V., Chyczy, M., Etschenberg, J. and Holstein, A.F. (1983) Induction of high titer mouse-antihuman spermatozoal antibodies by liposome incorporation of spermatozoal membrane antigens. Am. J. Reprod. Immunol. 4, 127-132. Mettler, L., Czuppon, A.B. and Tinneberg, H.-R. (1983) Immunization with spermatozoal peptide antigens resulting in immuno-suppression of fertility rates in female rats. Andrologia 15, 670-675. McLaren, A. (1964) Immunological control of fertility in female mice. Nature (London) 201, 582-585. Noyes, R.W., Odom, C.B. and Camyre, M.R. (1983) Radioisotope assay of sperm-binding globulin. Contraception 7, 57-59. Skrabei, H, Czuppon, A.B. and Mettler, L. (1980) Reaction of human spermatozoal haptens with sperm antibody containing sera of sterile males and females (protein-A tracer RIA) Arch. Androl. 5, 13-15.