Clin. exp. Immunol. (1977) 28, 223-225.
Reproducibility of the indirect immunofluorescence test on swollen human spermheads TALMA SAMUEL, NANCY J. ALEXANDER* & PH. ROMKE Division of Immunology, Antoni van Leeuwenhoekhuis, The Netherlands, Cancer Institute, Amsterdam, The Netherlands and * Oregon Regional Primate Research Center, Beaverton, Oregon, U.S.A.
(Received 8 December 1976) SUMMARY
Sera of 111 vasectomized men and infertile men and women were tested in each of two laboratories with the indirect immunofluorescence test on swollen human spermatozoa to detect antibodies against nuclear antigens specific to sperm. Comparison of the results revealed complete agreement in 110 of the sera. There were seventeen concordant positive sera (eight out of eighteen sera from vasectomized men, eight out of seventeen sera from infertile men, and one out of seventy-six infertile women). Most of the antibodies were of the IgG classs.
INTRODUCTION Some sera of infertile men contain antibodies that react specifically in the indirect immunofluorescence test with the nucleus of spermatozoa. This reaction requires artificially swollen spermatozoa (Kolk, Samuel & Rumke, 1974). Absorption experiments show that the sperm-specific nuclear antigens involved in the reaction are two strongly basic proteins, both with a mol. wt of about 6500 and a high arginine content, i.e., protamines (Kolk & Samuel, 1975). Antibodies to antigens of the sperm nucleus found in 22-33% of men 1 year after vasectomy were not present before vasectomy (Samuel et al., 1975; Tung, 1975). Vasectomized mice also developed antiprotamine antibodies, detected on human sw-ollen sperma-
(Samuel et al., 1976b). Since the titres of antiprotamine antibodies detected in this test are often very low, it seemed important to demonstrate in a co-operative study the reproducibility of such antibody detection. tozoa
MATERIALS AND METHODS Sperm. The swollen spermhead slides were prepared using a buffer composed of 50 mm Tris-HCl, 0 4 mm Dithiothreitol and 10 pg/ml trypsin at pH 9 0 as described earlier (Samuel et al., 1976a). Sera. The same 111 sera were tested in both laboratories. They were supplied by the WHO Reference Bank for Immunology of Reproduction in Aarhus, Denmark, and were mainly from vasectomized men and infertile men and women. All sera were coded, shipped in dry ice, and arrived frozen at both laboratories. The results of our studies were sent to the WHO Reference Bank in Aarhus, and subsequently the sera were decoded at a workshop held in Aarhus in August, 1976.
Indirect immunofluorescence test. In the Amsterdam laboratory (Lab A), the indirect immunofluorescence test (IFT) on swollen spermheads was performed as described earlier (Samuel et al., 1976a) with fluorescein-conjugated horse anti-total human immunoglobulin. The sera were screened undiluted, and positive sera were titrated in two-fold dilution series. IgA antibodies were detected with rabbit anti-human IgA antiserum and fluorescein conjugated horse anti-rabbit immunoglobulin antiserum. All antisera and conjugates were purchased from the Central Laboratory ofthe Blood Transfusion Service of the Netherlands Red Cross, Amsterdam, The Netherlands. The indirect IFT on swollen spermheads was performed in the Beaverton laboratory (Lab B) by screening at a 1:10 dilution. Fluorescein-conjugated rabbit anti-human IgG and IgM from Cappel Laboratories, Inc. (Downington, Pennsylvania, U.S.A.) were used. Positive sera were titred in a two-fold dilution series.
Correspondence: Dr T. Samuel, Division of Immunology. Antoni van Leeuwenhoekhuis, The Netherlands Cancer Institute, Sarphatistraat 108, Amsterdam, The Netherlands.
223
224
Talma Samuel, Nancy 7. Alexander & Ph. Rfimke
RESULTS A total of 111 sera, thirty-five from men (m) and seventy-six from women (f) were tested; eighty-nine (15 m, 74 f) of them were found negative in both laboratories. The results for the remaining twenty-two sera are presented in Table 1. Seventeen sera (16 m, 1 f) were found positive in both laboratories. Four (3 m, 1 f) were found positive by Lab A in titres lower than 10, Lab B, in which the sera were not tested in dilutions lower than 1:10 found these four negative in that dilution, thus agreeing with Lab A. The only discordant findings concerned one serum found negative in Lab A and positive with a titre of 10 in Lab B. Table 1 demonstrates the apparently good correlation between titres. TABLE 1. Comparison of the positive results obtained with the indirect IFT on swollen human spermheads in two laboratories
Lab A
Lab B
Serum source
Infertile men (17)
Vasectomized men (18)
Infertile women (76)
Serum code
Titre (total Ig)
Titre*
IgG
IgM
16 17 105 5 32 2 18 15 26 30 29
32 32 32 16 16 16 16
40 20 10 40 20 10 10 10
+ + + + + + + + -
-
65 77 28 99 97 101 21 11 95 9 86
-
8 4 2 10/17 64 64 64 32 16 16 8 8 8 9/18 4 2 2/76
-
10 -
9/17 40 40 20 40 20 10 10 10 -
8/18 10 -
-
± + + + + + + -
-
-
-
-
-
-
-
-
1/76
* Lowest dilution tested was 1:10.
After the results of Lab B were made known, all positive sera were retested in Lab A in a dilution of 1:2 for the presence of IgA antibodies. Only two sera (nos 16 and 28) were weakly positive. These two were then also retested for IgA antibodies in Lab B, where they were found negative at a dilution of 1:10.
DISCUSSION Antibodies to nuclear antigens can be detected by the indirect immunofluorescence test on swollen spermatozoa (Samuel et al., 1976a). If the reaction appears to take place only with spermatozoa, and not with other cells, the antibodies probably are directed against protamine. This was earlier shown in absorp-
Indirect IFT on swollen human spermheads
225
tion studies with purified human protamine (Kolk & Samuel, 1975). Antibodies to protamine, detected on swollen spermheads, were found in other studies (Samuel et al., 1975; Samuel et al., 1976b; Tung, 1975) after vasectomy but not before; moreover, they developed in general only when other antibodies to spermatozoa had developed. Detection of anti-protamine antibodies may thus indicate some form of autoimmunization against spermatozoa. Since titres were generally found to be low, some doubt remained as to the significance oftheir detection. The present study, however shows a clear correlation ofpositivity and titres for the same sera tested in two laboratories. Of the seventy-six sera from infertile women only two were positive, and only one of these two was found positive at a dilution of 1:10 in Lab B and 1:4 in Lab A. Moreover the two laboratories investigating sera for ANA both found this positive female serum to produce immunofluorescent staining of the nuclei of somatic target cells (WHO Workshop Report Aarhus, 1977). Thus, as in an earlier study (Kolk et al., 1974) antiprotamine antibodies were found to be unusual in sera of infertile women. This study confirms reports that the antibodies detected in this test in sera of infertile and vasectomized men are primarily of the IgG class (Kolk et al., 1974; Tung, 1975). Since a 99%/0 concurrence (110 out of 111 sera tested) was achieved in this comparative study we conclude that the indirect IFT on swollen human spermatozoa is a highly reproducible and reliable test for the detection of antibodies to nuclear antigens of spermatozoa. We gratefully acknowledge receipt of the sera used from the WHO Reference Bank for Reproductive Immunology, and wish to thank the anonymous laboratories which deposited these sera and the patients' records in the Bank. This study was supported pursuant to contract numbers NIH-NICHD-73-2700, and HD-4-2866 and grant number RR00163 with the National Institute of Health, Department of Health, Education and Welfare, U.S.A. This is publication No. 868 of the Oregon Regional Primate Research Center.
REFERENCES KOLK, A.H.J., SAMUEL, T. & RUMKE, P. (1974) Autoantigens of human spermatozoa. I. Solubilization of a new autoantigen detected on swollen spermheads. Clin. exp. Immunol. 16, 63. KOLK, A.H.J. & SAMUEL, T. (1975) Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa. Biochim. biophys. Acta (Amst.), 393, 307. SAMUEL, T., KOLK, A.H.J., RUMKE, P. & VAN Lis, J.M.J. (1975) Autoimmunity to sperm antigens in vasectomized men. Clin. exp. Immunol. 21, 65. SAMUEL, T., KOLK, A.H.J., RUMKE, P., AARDEN, L.A. & BUSTIN, M. (1976a) Histone and DNA detection in swollen spermatozoa and somatic cells, by immunofluorescence. Clin. exp. Immunol. 24, 63.
SAMUEL, T., CREWE, P.R., KOLK, A.H.J., RUiMKE, P. & BARNES, R.D. (1976b) Cross reaction of antibodies present in sera of vasectomized mice with human swollen spermheads. Clin. exp. Immunol. 25, 80. TUNG, K.S.K. (1975) Human sperm antigens and antisperm antibodies. I. Studies on vasectomy patients. Clin. exp. Immunol. 20, 93. WORKSHOP REPORT, AARHUS, (1977) Auto- and iso-antibodies to antigens of the human reproductive system. I. Results of an International Comparative Study of Antibodies to Spermatozoa and Other Antigens Detected in Sera from Infertile Patients Deposited in the WHO Reference Bankfor Reproductive Immunology, Aarhus, Denmark. (In press.)
Reproducibility of the indirect immunofluorescence test on swollen human spermheads TALMA SAMUEL, NANCY J. ALEXANDER* & PH. ROMKE Division of Immunology, Antoni van Leeuwenhoekhuis, The Netherlands, Cancer Institute, Amsterdam, The Netherlands and * Oregon Regional Primate Research Center, Beaverton, Oregon, U.S.A.
(Received 8 December 1976) SUMMARY
Sera of 111 vasectomized men and infertile men and women were tested in each of two laboratories with the indirect immunofluorescence test on swollen human spermatozoa to detect antibodies against nuclear antigens specific to sperm. Comparison of the results revealed complete agreement in 110 of the sera. There were seventeen concordant positive sera (eight out of eighteen sera from vasectomized men, eight out of seventeen sera from infertile men, and one out of seventy-six infertile women). Most of the antibodies were of the IgG classs.
INTRODUCTION Some sera of infertile men contain antibodies that react specifically in the indirect immunofluorescence test with the nucleus of spermatozoa. This reaction requires artificially swollen spermatozoa (Kolk, Samuel & Rumke, 1974). Absorption experiments show that the sperm-specific nuclear antigens involved in the reaction are two strongly basic proteins, both with a mol. wt of about 6500 and a high arginine content, i.e., protamines (Kolk & Samuel, 1975). Antibodies to antigens of the sperm nucleus found in 22-33% of men 1 year after vasectomy were not present before vasectomy (Samuel et al., 1975; Tung, 1975). Vasectomized mice also developed antiprotamine antibodies, detected on human sw-ollen sperma-
(Samuel et al., 1976b). Since the titres of antiprotamine antibodies detected in this test are often very low, it seemed important to demonstrate in a co-operative study the reproducibility of such antibody detection. tozoa
MATERIALS AND METHODS Sperm. The swollen spermhead slides were prepared using a buffer composed of 50 mm Tris-HCl, 0 4 mm Dithiothreitol and 10 pg/ml trypsin at pH 9 0 as described earlier (Samuel et al., 1976a). Sera. The same 111 sera were tested in both laboratories. They were supplied by the WHO Reference Bank for Immunology of Reproduction in Aarhus, Denmark, and were mainly from vasectomized men and infertile men and women. All sera were coded, shipped in dry ice, and arrived frozen at both laboratories. The results of our studies were sent to the WHO Reference Bank in Aarhus, and subsequently the sera were decoded at a workshop held in Aarhus in August, 1976.
Indirect immunofluorescence test. In the Amsterdam laboratory (Lab A), the indirect immunofluorescence test (IFT) on swollen spermheads was performed as described earlier (Samuel et al., 1976a) with fluorescein-conjugated horse anti-total human immunoglobulin. The sera were screened undiluted, and positive sera were titrated in two-fold dilution series. IgA antibodies were detected with rabbit anti-human IgA antiserum and fluorescein conjugated horse anti-rabbit immunoglobulin antiserum. All antisera and conjugates were purchased from the Central Laboratory ofthe Blood Transfusion Service of the Netherlands Red Cross, Amsterdam, The Netherlands. The indirect IFT on swollen spermheads was performed in the Beaverton laboratory (Lab B) by screening at a 1:10 dilution. Fluorescein-conjugated rabbit anti-human IgG and IgM from Cappel Laboratories, Inc. (Downington, Pennsylvania, U.S.A.) were used. Positive sera were titred in a two-fold dilution series.
Correspondence: Dr T. Samuel, Division of Immunology. Antoni van Leeuwenhoekhuis, The Netherlands Cancer Institute, Sarphatistraat 108, Amsterdam, The Netherlands.
223
224
Talma Samuel, Nancy 7. Alexander & Ph. Rfimke
RESULTS A total of 111 sera, thirty-five from men (m) and seventy-six from women (f) were tested; eighty-nine (15 m, 74 f) of them were found negative in both laboratories. The results for the remaining twenty-two sera are presented in Table 1. Seventeen sera (16 m, 1 f) were found positive in both laboratories. Four (3 m, 1 f) were found positive by Lab A in titres lower than 10, Lab B, in which the sera were not tested in dilutions lower than 1:10 found these four negative in that dilution, thus agreeing with Lab A. The only discordant findings concerned one serum found negative in Lab A and positive with a titre of 10 in Lab B. Table 1 demonstrates the apparently good correlation between titres. TABLE 1. Comparison of the positive results obtained with the indirect IFT on swollen human spermheads in two laboratories
Lab A
Lab B
Serum source
Infertile men (17)
Vasectomized men (18)
Infertile women (76)
Serum code
Titre (total Ig)
Titre*
IgG
IgM
16 17 105 5 32 2 18 15 26 30 29
32 32 32 16 16 16 16
40 20 10 40 20 10 10 10
+ + + + + + + + -
-
65 77 28 99 97 101 21 11 95 9 86
-
8 4 2 10/17 64 64 64 32 16 16 8 8 8 9/18 4 2 2/76
-
10 -
9/17 40 40 20 40 20 10 10 10 -
8/18 10 -
-
± + + + + + + -
-
-
-
-
-
-
-
-
1/76
* Lowest dilution tested was 1:10.
After the results of Lab B were made known, all positive sera were retested in Lab A in a dilution of 1:2 for the presence of IgA antibodies. Only two sera (nos 16 and 28) were weakly positive. These two were then also retested for IgA antibodies in Lab B, where they were found negative at a dilution of 1:10.
DISCUSSION Antibodies to nuclear antigens can be detected by the indirect immunofluorescence test on swollen spermatozoa (Samuel et al., 1976a). If the reaction appears to take place only with spermatozoa, and not with other cells, the antibodies probably are directed against protamine. This was earlier shown in absorp-
Indirect IFT on swollen human spermheads
225
tion studies with purified human protamine (Kolk & Samuel, 1975). Antibodies to protamine, detected on swollen spermheads, were found in other studies (Samuel et al., 1975; Samuel et al., 1976b; Tung, 1975) after vasectomy but not before; moreover, they developed in general only when other antibodies to spermatozoa had developed. Detection of anti-protamine antibodies may thus indicate some form of autoimmunization against spermatozoa. Since titres were generally found to be low, some doubt remained as to the significance oftheir detection. The present study, however shows a clear correlation ofpositivity and titres for the same sera tested in two laboratories. Of the seventy-six sera from infertile women only two were positive, and only one of these two was found positive at a dilution of 1:10 in Lab B and 1:4 in Lab A. Moreover the two laboratories investigating sera for ANA both found this positive female serum to produce immunofluorescent staining of the nuclei of somatic target cells (WHO Workshop Report Aarhus, 1977). Thus, as in an earlier study (Kolk et al., 1974) antiprotamine antibodies were found to be unusual in sera of infertile women. This study confirms reports that the antibodies detected in this test in sera of infertile and vasectomized men are primarily of the IgG class (Kolk et al., 1974; Tung, 1975). Since a 99%/0 concurrence (110 out of 111 sera tested) was achieved in this comparative study we conclude that the indirect IFT on swollen human spermatozoa is a highly reproducible and reliable test for the detection of antibodies to nuclear antigens of spermatozoa. We gratefully acknowledge receipt of the sera used from the WHO Reference Bank for Reproductive Immunology, and wish to thank the anonymous laboratories which deposited these sera and the patients' records in the Bank. This study was supported pursuant to contract numbers NIH-NICHD-73-2700, and HD-4-2866 and grant number RR00163 with the National Institute of Health, Department of Health, Education and Welfare, U.S.A. This is publication No. 868 of the Oregon Regional Primate Research Center.
REFERENCES KOLK, A.H.J., SAMUEL, T. & RUMKE, P. (1974) Autoantigens of human spermatozoa. I. Solubilization of a new autoantigen detected on swollen spermheads. Clin. exp. Immunol. 16, 63. KOLK, A.H.J. & SAMUEL, T. (1975) Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa. Biochim. biophys. Acta (Amst.), 393, 307. SAMUEL, T., KOLK, A.H.J., RUMKE, P. & VAN Lis, J.M.J. (1975) Autoimmunity to sperm antigens in vasectomized men. Clin. exp. Immunol. 21, 65. SAMUEL, T., KOLK, A.H.J., RUMKE, P., AARDEN, L.A. & BUSTIN, M. (1976a) Histone and DNA detection in swollen spermatozoa and somatic cells, by immunofluorescence. Clin. exp. Immunol. 24, 63.
SAMUEL, T., CREWE, P.R., KOLK, A.H.J., RUiMKE, P. & BARNES, R.D. (1976b) Cross reaction of antibodies present in sera of vasectomized mice with human swollen spermheads. Clin. exp. Immunol. 25, 80. TUNG, K.S.K. (1975) Human sperm antigens and antisperm antibodies. I. Studies on vasectomy patients. Clin. exp. Immunol. 20, 93. WORKSHOP REPORT, AARHUS, (1977) Auto- and iso-antibodies to antigens of the human reproductive system. I. Results of an International Comparative Study of Antibodies to Spermatozoa and Other Antigens Detected in Sera from Infertile Patients Deposited in the WHO Reference Bankfor Reproductive Immunology, Aarhus, Denmark. (In press.)
