- Email: [email protected]
Detection of anti-sperm activities of monoclonal antibodies to human sperm
Journal of Reproductive Immunology, 7 (1985) 215-223 Elsevier
215
JRI 00348
Detection of anti-sperm activities of monoclonal antibodies to human sperm R.J.T. Hancock * and S. Faruki Division of lmmunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London N W 7 1AA, U.K. (Accepted for publication 14 December 1984)
The anti-sperm activities of a series of monoclonal antibodies to human sperm have been compared using agglutination, immunofluorescence, ELISA and 'panning' assays. The antibodies fell into two categories, those that could be detected by agglutination but not immunofluorescence assays and those that could be detected by immunofluorescence but not agglutination. Antibodies positive in the agglutination assays were also positive in the 'panning' assay. None of the antibodies tested was positive in the ELISA assays. These results, and others, are discussed in relation to the problems associated with the detection of anti-sperm antibodies in sub-fertile human populations.
Key words: human sperm monoclonals, agglutination, immunofluorescence, panning and ELISA assays
Introduction
Antibodies to sperm apparently contribute to subfertility in human populations (e.g. Menge, 1980; Hancock, 1981). Characterization of sperm components using monoclonal antibodies (Mabs) might provide some insight into the nature of the antigens with which human anti-sperm antibodies react and a number of laboratories have produced Mabs to human sperm components (e.g. Lee et al., 1982; Menge et al., 1983; Wolf et al., 1983; Hancock and Faruki, unpubl, data). In our own investigations we observed very striking differences between the anti-sperm activities of different Mabs when assayed by different procedures. These observations are relevant to some of the problems associated with the clinical detection of anti-sperm antibodies in human populations and are therefore described and discussed in detail in this report.
* Present address: U.K. Transplant Service, South Western Regional Transfusion Centre, Southmead, Bristol BS10 5ND, U.K. 0165-0378/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
216
Materials and Methods
Sperm Human sperm provided by the Seminology Laboratory at Chelsea Hospital for Women were stored frozen in egg yolk diluent (Richardson, 1976) until used in the assays for anti-sperm activity. Sperm used for immunizing mice were not exposed to egg yolk diluent. Ejaculated rabbit sperm were obtained using an artificial vagina (Hafez, 1970) and mouse sperm were obtained by chopping up the epididymes in PBS and drawing off the sperm in the supernatant.
Monoclonal antibodies (Mabs) These were produced essentially as described by Fazekas de St. Groth and Scheidegger (1980). Immune spleen cells from a BALB/c mouse immunized with washed whole human sperm were fused with P3 x 63Ag8653 myeloma cells, the hybridomas screened for anti-sperm antibodies by agglutination or immunofluorescence assays (see below) and cloned once or twice by limiting dilution. No substantial investigations were carried out to determine whether differences in reaction patterns might have been observed if antibody testing had been carried out using fresh semen rather than frozen sperm. Isotype subclasses of the Mabs were determined by immunodiffusion in 1% agar against class-specific antisera (Litton Bionetics, Charleston, SC, U.S.A.; Miles Laboratories, Slough, England). The hybridoma lines are referred to by numbers (10.1, 10.2 etc.) which are abbreviations of NIM 10.1, NIM 10.2 etc., their official reference numbers.
Indirect immunofluorescence assay Sperm were washed twice in PBS (centrifuging at 550 x g for 5 min), resuspended in PBS to 1-2 x 106/ml and sperm from 0.1 ml vol. cytocentrifuged onto clean microscope slides (20 x g for 5 min). 20/~1 drops of test supernatant were added to the sperm and incubated in a humidified container for 20 min at 4°C. The slides were then rinsed three times in PBS, 20 /~1 of FITC-conjugated rabbit anti-mouse immunoglobulin (Miles Laboratories, Slough, England) were added and the slides incubated for a further 20 min at 4°C. The slides were washed three times in PBS, the cells mounted in 20/~1 of 50% glycerol in PBS, the coverslip edges sealed with nail varnish and the slides examined on a Leitz Orthoplan microscope.
Sperm agglutination assay The procedure was based on the tray agglutination test described by Friberg (1974) for the detection of human anti-sperm antibodies. Semen was thawed, diluted to 20 x 106/ml and equal volumes of sperm and test supernatant mixed. 5/xl drops of the mixture were pipetted into oil-filled tissue-typing trays (Hamax A.S., Moss, Norway) and examined at intervals using an inverted microscope.
Immunofluorescence assay for anti-human leucocyte activity Human peripheral blood leucocytes prepared essentially as described by Birkeland (1980) were washed twice in PBS, centrifuging at 180 x g for 5 min and
217
resuspended at 2 × 106/ml. 0.1 ml aliquots were mixed with 20 /~1 vols. of tissue culture supernatant, incubated at 4°C for 20 min, washed twice in PBS, resuspended in 20 #1 FITC-conjugated rabbit anti-mouse immunoglobulin, and incubated for a further 20 min at 4°C. The cells were then washed twice in PBS, the cells resuspended in 20 #1 of 50% glycerol in PBS and mounted as before. Any cell lines which showed anti-leucocyte activity were not studied further.
'Panning' assay for anti-sperm antibodies This was carried out essentially as described previously (Hancock and Faruki, 1984). The essentials of the method are that sperm are incubated in solutions of anti-sperm antibody, washed and incubated in wells coated with anti-immunoglobulin molecules. The wells are then washed and examined to determine whether the presence of anti-sperm antibody on the sperm has caused the sperm to stick to the wells coated with anti-immunoglobulin molecules (see Fig. 1). Among the advantages of this procedure are that it does not involve sperm fixation and can be used with sperm of low motility. The plates can be stained and the photographs presented are of fixed stained sperm.
ELISA assay A procedure based on methods described by Hudson and Hay (1980), Witkin et Sperm
4. Anti-Sperm Antibody
1
4. Well Specifically Coated
with Anti-lmmunoglobulin Molecules
r
Fig. 1. Diagrammatic s u m m a r y of ' p a n n i n g ' assay for anti-sperm antibodies. Sperm are incubated in solutions of anti-sperm antibody, washed and incubated in wells coated with anti-immunoglobulin molecules. The wells are then washed and examined to determine whether the presence of anti-sperm antibody on the sperm has caused the sperm to stick to the wells.
218 al. (1980, 1983) and Zanchetta et al. (1982) was used. Sperm were washed three times in PBS, adjusted to 5 x 106/ml, 50/tl vols. added to wells of plates (Dynatech) and 50 #1 of 0.25% glutaraldehyde added to bind the sperm to the bases. The plates were centrifuged at 250 x g for 5 min, the supernatant discarded and the target sperm used either 'unblocked' (following Zanchetta et al., 1982; Witkin, 1983) or after 'blocking' with 15% FCS. The wells were washed three times with PBS + 0.05% Tween 20 (Sigma), 50 /~1 of supernatant or diluted serum added and the plates incubated for 1 h at 37°C. The wells were then washed three times with PBS/Tween, 50/~1 of alkaline phosphatase conjugated rabbit anti-mouse immunoglobulin (Sigma) diluted 1/1000 in PBS/Tween added and the plates incubated for a further 1 h at 37°C. The wells were washed three times with PBS/Tween, 150 #1 substrate (phosphatase substrate tablets (Sigma) dissolved in diethanolamine buffer) were added, and the plates incubated at room temperature for 10-20 rain. The reactions were stopped by the addition of 50/~1 5N N a O H and the optical densities of the wells read on a Microelisa Minireader (Dynatech).
Results
When supernatants were screened by the sperm agglutination assay it was found that they could be conveniently divided into two categories: those that rapidly agglutinated the majority of the sperm into networks and those that did not. These differences were clear soon after mixing the sperm (10-20 rain) and were still clear after several hours. Three cell lines (10.09, 10.10, 10.11) producing this pattern were selected after cloning once or twice. No evidence was obtained on whether the three sperm agglutinating Mabs were directed against the same or different antigens. Interestingly, none of these antibodies was positive in the immunofluorescence assay. When supernatants were screened by immunofluorescence a variety of positive patterns were observed and 8 cell lines (10.1-10.8) were grown up after cloning once or twice. Under the experimental conditions used there was no substantial nonspecific binding of immunoglobulin from control normal mouse serum or control Mabs directed against non-sperm antigens. Cell lines producing antibodies which showed cross-reactivity with human somatic cells (see Materials and Methods) were not studied further. The Mabs detected by immunofluorescence were not apparently all directed against the same antigen since a variety of staining patterns were observed, e.g. Mab 10.2 stained the whole cell brightly while 10.3 stained the tail preferentially and 10.1 the tail and rear of the head. The antigens with which these Mabs reacted also showed some differences in the degree to which they were removed by detergent treatment of target sperm (Table 1) but they were generally apparently relatively insoluble. They were also not removed by treating the cytocentrifuged sperm with 95% chloroform/5% methanol before adding the Mabs. The antigens with which Mabs 10.1, 10.4, 10.6 reacted were largely removed by treating the cytocentrifuged sperm with 0.25% trypsin for 30 rain at 37°C before stopping the reaction with 0.1 mM phenylmethylsulfonyl fluoride (Sigma), washing the cells and adding the Mabs. The antigens with which the other Mabs reacted were affected less
219 TABLE 1 Effects of detergent treatment on antigens with which MoAbs 10.1-10.8 react Detergent
MoAbs 10.1
1% NP40 0.01% SDS 0.1% SDS 0.1% Triton 1.0% Triton
. . + . .
10.2
10.3
.
.
.
.
.
. +
+ . .
10.4
. .
10.5
.
.
.
.
. .
+ . .
10.7
10.8
+ +
+ +
.
. +
. .
10.6
+ . .
. .
Cytocentrifuged sperm were treated with the above detergents for 30 min and the effects on their subsequent reactivity with MoAbs determined by immunofluorescence. + , Antigenicity decreased; + , antigenicity partly decreased; - , antigenicity not decreased.
by this treatment. The Mabs did not show any reactions with mouse or rabbit sperm when tested by immunofluorescence. None of the antibodies produced by cell lines 10.1-10.8 showed obvious sperm agglutinating activity (see summary Table 3). When the Mabs were tested by panning they fell into two categories: those that caused attachment to the panning plates in very large numbers (see Fig. 2a) and those that did not produce these effects (Fig. 2b). The Mabs positive in this assay were the same ones that were positive in the agglutination assay (see s u m m a r y Table 3).
TABLE 2 Anti-sperm activities of MoAb supernatants and control sera assessed by ELISA assay Solutions
Optical densities (SE)
tested
' Unblocked' target sperm
Target s p e r m ' blocked' with 15% FCS
1.12 (0.13)
0.70 (0.12)
0.69 0.04 0.03 0.03 0.05 0.05 0.08 0.06 0.09 0.06 0.04 0.04
0.46 0.00 0.04 0.07 0.01 0.03 0.04 0.10 0.12 0.06 0.05 0.02
Mouse anti-human sperm serum, 1 / 4 0 Normal mouse serum, 1 / 4 0 M o A b 10.1 M o A b 10.2 M o A b 10.3 M o A b 10.4 M o A b 10.5 M o A b 10.6 M o A b 10.7 MoAb 10.8 MoAb 10.9 MoAb 10.10 M o A b 10.11
(0.04) (0.01) (0.01) (0.01) (0.00) (0.02) (0.01) (0.01) (0.03) (0.02) (0.01) (0.01)
(0.05) (0.00) (0.01) (0.01) (0.01) (0.01) (0.01) (0.04) (0.05) (0.01) (0.03) (0.01)
Assay carried out as described in text. All MoAb supernatants showed less anti-sperm activity than 1 / 4 0 dilution of normal mouse serum.
220
b Fig. 2. (a) Human sperm treated with MoAb 10.11 bound to 'panning' plate in large numbers ( × 285). (b) Human sperm treated with MoAb 10.2 showing no substantial binding to 'panning' plate ( × 285).
221
TABLE 3 S u m m a r y o f a n t i - s p e r m activities of M o A b s a s s a y e d b y d i f f e r e n t p r o c e d u r e s Assays
Immunofluorescence Agglutination ' Panning' ELISA
Monoclonal antibodies 10.1
10.2
10.3
10.4
10.5
10.6
10.7
10.8
10.9
10.10
10.11
+ . .
+
+
+ .
+
+
+
+
+ +
+ +
+ +
.
.
.
.
.
. .
. .
. .
. . .
. .
. .
.
.
.
A n t i b o d y class: M o A b s 1 0 . 1 - 1 0 . 8 , I g G l ; M o A b s 1 0 . 9 - 1 0 . 1 1 , I g M .
None of the Mabs was positive in the ELISA assay described. All showed less binding to the glutaraldehyde fixed sperm than did the immunoglobulin in the normal mouse serum negative control (Table 2). This was observed with both 'unblocked' target sperm and target sperm blocked with 15% FCS. The Mabs similarly failed to show anti-sperm activity when sperm treated with 0.5% glutaraldehyde (Witkin, 1983) were used as targets (data not shown). The antibody classes of the Mabs are given in Table 3.
Discussion
The results reported in this paper clearly demonstrated that different sperm-antibody interactions were detected with differing sensitivities by the different assays (see summary Table 3). The results reported have obvious important practical implications since they emphasize that one should be cautious in assuming that the anti-sperm activities detected in sera by different assays are in fact measuring the same interactions. This applies to all new procedures but probably particularly to the ELISA procedures because their apparently greater convenience (compared with sperm agglutination) may tempt inexperienced workers to use such procedures uncritically for the clinical assessment of anti-sperm immunity. The reasons for some of the differences reported are not obvious, but appear scientifically interesting and are under further investigation. It is not surprising that IgM antibodies should be more readily detectable by an agglutination assay than IgG, but the reasons for the failure to detect the anti-sperm activities of Mabs 10.9, 10.10 and 10.11 by immunofluorescence are less clear. One hypothesis would be that the antigens with which these Mabs react are present at a low density on the membrane, sufficient to allow agglutination but insufficient to produce detectable immunofluorescence. An alternative hypothesis would be that the molecules are lost from the sperm or lose their antigenic conformation when the sperm are cytocentrifuged and repeatedly washed. There is no reason to suppose that the failure to detect the activity was caused by inability of the FITC-conjugated anti-mouse immunoglobulin to bind to IgM since the anti-immunoglobulin was directed against both heavy and light chains and reacted with IgM conjugated to Sepharose beads. In
222 addition, a specific FITC-conjugated anti-IgM (a generous gift from R.M.E. Parkhouse) failed to show any convincing fluorescence with these Mabs on sperm. Discrepancies between the results of immunofluorescence and sperm agglutination assays have also been reported when these procedures were used to test human sera (e.g. Menge, 1980; Hancock, 1981). The observation that the Mabs which showed strong sperm-agglutinating activity also showed very striking positive reactions in the panning procedure while the Mabs negative in the agglutination assay did not (see Table 3) was also interesting. Whether this occurred because of differences between the antigens with which the different Mabs were reacting or solely because of differences in immunoglobulin class cannot be readily determined. There is, however, evidence that the panning assay is capable of detecting anti-sperm IgG (Hancock and Faruki, 1984) which would, to a degree, argue against the second hypothesis. One could speculate that the panning assay might be more sensitive for more superficial antigens (which would also be detected in agglutination assays) and be less sensitive for less superficial antigens which might become exposed, and therefore detected, by the immunofluorescence procedure. The observation that the antigens reacting with Mabs 10.1-10.8 were not removed by Triton treatment (Table 1) would be consistent with their being less superficial antigens (Jones et al., 1983). Although anti-sperm activity could be detected in mouse anti-human sperm serum (see Table 2) using the ELISA procedure described, this assay clearly did not demonstrate anti-sperm activity in the supernatants tested. In fact, the normal mouse serum negative controls showed substantially more anti-sperm activity than the Mab supernatants. This was not observed with any of the other assays used. The binding of immunoglobulin from normal mouse serum to sperm was increased when the sperm were treated with glutaraldehyde (data not shown - tested by immunofluorescence). Whether the binding of immunoglobulin from normal mouse serum to glutaraldehyde-treated sperm in the ELISA assay described reflects the binding of 'natural' anti-sperm antibodies (see Hancock, 1981, 1984 for recent reviews) or non-specific binding of immunoglobulin via the Fc part of the molecule (e.g. Witkin et al., 1980) was not determined. However, whatever the basis, the phenomenon is worth bearing in mind when considering the use of glutaraldehyde for treating target sperm. It clearly makes the sperm unsuitable for the assessment of some antigen-antibody interactions. In summary, we have found that different sperm-monoclonal antibody interactions were detected with differing sensitivities by different assays. These results have obvious practical implications and the biological reasons for them are under further investigation.
Acknowledgements We would like to thank our colleagues, particularly G. Preece and M. Kennedy, for their help and advice, D. Wraith for reading the manuscript and M. Bertagne for typing it. We thank W.F. Hendry, J. Stedronska and C. Stock for arranging for the supplies of human sperm from the Seminology Laboratory at Chelsea Hospital.
223 References Birkeland, S.A. (1980) Cryopreservation of human lymphocytes for sequential testing of immune competence. J. Immunol. Methods 35, 57-67. Fazekas de St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1-21. Friberg, J. (1974) Clinical and immunological studies on sperm-agglutinating antibodies in serum and seminal plasma. Acta Obstet. Gynaecol. Scand., Suppl. 36, 3-76. Hafez, E.S.E. (1970) Rabbits. In Reproduction and Breeding Techniques for Laboratory Animals (Hafez, E.S.E., ed.) pp. 273-298. Lea and Febiger, Philadelphia. Hancock, R.J.T. (1981) Immune responses to sperm. In Oxford Reviews of Reproductive Biology III (Finn, C.A., ed.), pp. 183-208. Clarendon Press, Oxford. Hancock, R.J.T. (1984) Immune responses to sperm - recent developments. In Proceedings of 38th Sutton Bonnington Easter School: Immunological Aspects of Reproduction in Mammals (Crighton, D.B., ed.), pp. 55-76. Butterworths, London. Hancock, R.J.T. and Faruki, S. (1984) Detection of antibody-coated sperm by 'panning' procedures. J. Immunol. Methods 66, 149-159. Hudson, L. and Hay, F.C. (1980) Practical Immunology, Second Edition. Blackwell Scientific Publications, Oxford, p. 315. Jones, R., Brown, C.R., Cran, D.G. and Gaunt, S.S. (1983) Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies. Gamete Res. 8, 255-265. Lee, C.G., Huang, Y., Huang, C., Hu, P.C. and Menge, A.C. (1982) Monoclonal antibodies to human sperm antigens. J. Reprod. Immunol. 4, 173-181. Menge, A.C. (1980) Clinical immunologic infertility: diagnostic measures, incidence of antisperm antibodies, fertility and mechanisms. In Immunological Aspects of Fertility Regulation (Dhindsa, D.S. and Schumacher, F.B., eds.), pp. 205-224. Elsevier/North-Holland, Amsterdam. Menge, A.C., Richter, D.E., Naz, R.K., Lee, C.G. and Wong, E. (1983) Dissecting the sperm cell by use of monoclonal antibodies. In Reproductive Immunology 1983 (Isojima, S. and Billington, W.D., eds.), pp. 81-90 Elsevier Science Publishers, Amsterdam. Richardson, D.W. (1976) Techniques of sperm storage. In Artificial Insemination: Proceedings of the 4th Study Group of the Royal College of Obstetricians and Gynaecologists (Brudenell, M., McLaren, A., Short, R.V. and Symonds, M., eds.), pp. 97-106. Royal College of Obstetricians and Gynaecologists, London. Witkin, S.S. (1983) Enzyme linked immunosorbent assay (ELISA) for detection of antibodies to spermatozoa. Res. Reprod. 15, 1. Witkin, S.S., Shahani, S.K., Gupta, S., Good, R.A. and Day, N.K. (1980) Demonstration of IgG Fc receptors on spermatozoa and their utilization for the detection of circulating complexes in human serum. Clin. Exp. Immunol. 41,441-452. Witkin, S.S., Zelikovsky, G., Good, R.A. and Day, N.K. (1981) Demonstration of llSIgA antibody to spermatozoa in human seminal fluid. Clin. Exp. Immunol. 44, 368-374. Wolf, D.P., Sokoloski, J.E., Dandekar, P. and Bechtol, K.B. (1983) Characterization of human sperm surface antigens with monoclonal antibodies. Biol. Reprod. 29, 713-723. Zanchetta, R., Busolo, F. and Mastrogiacomo, I. (1982) The enzyme-linked immunosorbent assay for detection of the anti-spermatozoal antibodies. Fertil. Steril. 38, 730-734.
215
JRI 00348
Detection of anti-sperm activities of monoclonal antibodies to human sperm R.J.T. Hancock * and S. Faruki Division of lmmunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London N W 7 1AA, U.K. (Accepted for publication 14 December 1984)
The anti-sperm activities of a series of monoclonal antibodies to human sperm have been compared using agglutination, immunofluorescence, ELISA and 'panning' assays. The antibodies fell into two categories, those that could be detected by agglutination but not immunofluorescence assays and those that could be detected by immunofluorescence but not agglutination. Antibodies positive in the agglutination assays were also positive in the 'panning' assay. None of the antibodies tested was positive in the ELISA assays. These results, and others, are discussed in relation to the problems associated with the detection of anti-sperm antibodies in sub-fertile human populations.
Key words: human sperm monoclonals, agglutination, immunofluorescence, panning and ELISA assays
Introduction
Antibodies to sperm apparently contribute to subfertility in human populations (e.g. Menge, 1980; Hancock, 1981). Characterization of sperm components using monoclonal antibodies (Mabs) might provide some insight into the nature of the antigens with which human anti-sperm antibodies react and a number of laboratories have produced Mabs to human sperm components (e.g. Lee et al., 1982; Menge et al., 1983; Wolf et al., 1983; Hancock and Faruki, unpubl, data). In our own investigations we observed very striking differences between the anti-sperm activities of different Mabs when assayed by different procedures. These observations are relevant to some of the problems associated with the clinical detection of anti-sperm antibodies in human populations and are therefore described and discussed in detail in this report.
* Present address: U.K. Transplant Service, South Western Regional Transfusion Centre, Southmead, Bristol BS10 5ND, U.K. 0165-0378/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
216
Materials and Methods
Sperm Human sperm provided by the Seminology Laboratory at Chelsea Hospital for Women were stored frozen in egg yolk diluent (Richardson, 1976) until used in the assays for anti-sperm activity. Sperm used for immunizing mice were not exposed to egg yolk diluent. Ejaculated rabbit sperm were obtained using an artificial vagina (Hafez, 1970) and mouse sperm were obtained by chopping up the epididymes in PBS and drawing off the sperm in the supernatant.
Monoclonal antibodies (Mabs) These were produced essentially as described by Fazekas de St. Groth and Scheidegger (1980). Immune spleen cells from a BALB/c mouse immunized with washed whole human sperm were fused with P3 x 63Ag8653 myeloma cells, the hybridomas screened for anti-sperm antibodies by agglutination or immunofluorescence assays (see below) and cloned once or twice by limiting dilution. No substantial investigations were carried out to determine whether differences in reaction patterns might have been observed if antibody testing had been carried out using fresh semen rather than frozen sperm. Isotype subclasses of the Mabs were determined by immunodiffusion in 1% agar against class-specific antisera (Litton Bionetics, Charleston, SC, U.S.A.; Miles Laboratories, Slough, England). The hybridoma lines are referred to by numbers (10.1, 10.2 etc.) which are abbreviations of NIM 10.1, NIM 10.2 etc., their official reference numbers.
Indirect immunofluorescence assay Sperm were washed twice in PBS (centrifuging at 550 x g for 5 min), resuspended in PBS to 1-2 x 106/ml and sperm from 0.1 ml vol. cytocentrifuged onto clean microscope slides (20 x g for 5 min). 20/~1 drops of test supernatant were added to the sperm and incubated in a humidified container for 20 min at 4°C. The slides were then rinsed three times in PBS, 20 /~1 of FITC-conjugated rabbit anti-mouse immunoglobulin (Miles Laboratories, Slough, England) were added and the slides incubated for a further 20 min at 4°C. The slides were washed three times in PBS, the cells mounted in 20/~1 of 50% glycerol in PBS, the coverslip edges sealed with nail varnish and the slides examined on a Leitz Orthoplan microscope.
Sperm agglutination assay The procedure was based on the tray agglutination test described by Friberg (1974) for the detection of human anti-sperm antibodies. Semen was thawed, diluted to 20 x 106/ml and equal volumes of sperm and test supernatant mixed. 5/xl drops of the mixture were pipetted into oil-filled tissue-typing trays (Hamax A.S., Moss, Norway) and examined at intervals using an inverted microscope.
Immunofluorescence assay for anti-human leucocyte activity Human peripheral blood leucocytes prepared essentially as described by Birkeland (1980) were washed twice in PBS, centrifuging at 180 x g for 5 min and
217
resuspended at 2 × 106/ml. 0.1 ml aliquots were mixed with 20 /~1 vols. of tissue culture supernatant, incubated at 4°C for 20 min, washed twice in PBS, resuspended in 20 #1 FITC-conjugated rabbit anti-mouse immunoglobulin, and incubated for a further 20 min at 4°C. The cells were then washed twice in PBS, the cells resuspended in 20 #1 of 50% glycerol in PBS and mounted as before. Any cell lines which showed anti-leucocyte activity were not studied further.
'Panning' assay for anti-sperm antibodies This was carried out essentially as described previously (Hancock and Faruki, 1984). The essentials of the method are that sperm are incubated in solutions of anti-sperm antibody, washed and incubated in wells coated with anti-immunoglobulin molecules. The wells are then washed and examined to determine whether the presence of anti-sperm antibody on the sperm has caused the sperm to stick to the wells coated with anti-immunoglobulin molecules (see Fig. 1). Among the advantages of this procedure are that it does not involve sperm fixation and can be used with sperm of low motility. The plates can be stained and the photographs presented are of fixed stained sperm.
ELISA assay A procedure based on methods described by Hudson and Hay (1980), Witkin et Sperm
4. Anti-Sperm Antibody
1
4. Well Specifically Coated
with Anti-lmmunoglobulin Molecules
r
Fig. 1. Diagrammatic s u m m a r y of ' p a n n i n g ' assay for anti-sperm antibodies. Sperm are incubated in solutions of anti-sperm antibody, washed and incubated in wells coated with anti-immunoglobulin molecules. The wells are then washed and examined to determine whether the presence of anti-sperm antibody on the sperm has caused the sperm to stick to the wells.
218 al. (1980, 1983) and Zanchetta et al. (1982) was used. Sperm were washed three times in PBS, adjusted to 5 x 106/ml, 50/tl vols. added to wells of plates (Dynatech) and 50 #1 of 0.25% glutaraldehyde added to bind the sperm to the bases. The plates were centrifuged at 250 x g for 5 min, the supernatant discarded and the target sperm used either 'unblocked' (following Zanchetta et al., 1982; Witkin, 1983) or after 'blocking' with 15% FCS. The wells were washed three times with PBS + 0.05% Tween 20 (Sigma), 50 /~1 of supernatant or diluted serum added and the plates incubated for 1 h at 37°C. The wells were then washed three times with PBS/Tween, 50/~1 of alkaline phosphatase conjugated rabbit anti-mouse immunoglobulin (Sigma) diluted 1/1000 in PBS/Tween added and the plates incubated for a further 1 h at 37°C. The wells were washed three times with PBS/Tween, 150 #1 substrate (phosphatase substrate tablets (Sigma) dissolved in diethanolamine buffer) were added, and the plates incubated at room temperature for 10-20 rain. The reactions were stopped by the addition of 50/~1 5N N a O H and the optical densities of the wells read on a Microelisa Minireader (Dynatech).
Results
When supernatants were screened by the sperm agglutination assay it was found that they could be conveniently divided into two categories: those that rapidly agglutinated the majority of the sperm into networks and those that did not. These differences were clear soon after mixing the sperm (10-20 rain) and were still clear after several hours. Three cell lines (10.09, 10.10, 10.11) producing this pattern were selected after cloning once or twice. No evidence was obtained on whether the three sperm agglutinating Mabs were directed against the same or different antigens. Interestingly, none of these antibodies was positive in the immunofluorescence assay. When supernatants were screened by immunofluorescence a variety of positive patterns were observed and 8 cell lines (10.1-10.8) were grown up after cloning once or twice. Under the experimental conditions used there was no substantial nonspecific binding of immunoglobulin from control normal mouse serum or control Mabs directed against non-sperm antigens. Cell lines producing antibodies which showed cross-reactivity with human somatic cells (see Materials and Methods) were not studied further. The Mabs detected by immunofluorescence were not apparently all directed against the same antigen since a variety of staining patterns were observed, e.g. Mab 10.2 stained the whole cell brightly while 10.3 stained the tail preferentially and 10.1 the tail and rear of the head. The antigens with which these Mabs reacted also showed some differences in the degree to which they were removed by detergent treatment of target sperm (Table 1) but they were generally apparently relatively insoluble. They were also not removed by treating the cytocentrifuged sperm with 95% chloroform/5% methanol before adding the Mabs. The antigens with which Mabs 10.1, 10.4, 10.6 reacted were largely removed by treating the cytocentrifuged sperm with 0.25% trypsin for 30 rain at 37°C before stopping the reaction with 0.1 mM phenylmethylsulfonyl fluoride (Sigma), washing the cells and adding the Mabs. The antigens with which the other Mabs reacted were affected less
219 TABLE 1 Effects of detergent treatment on antigens with which MoAbs 10.1-10.8 react Detergent
MoAbs 10.1
1% NP40 0.01% SDS 0.1% SDS 0.1% Triton 1.0% Triton
. . + . .
10.2
10.3
.
.
.
.
.
. +
+ . .
10.4
. .
10.5
.
.
.
.
. .
+ . .
10.7
10.8
+ +
+ +
.
. +
. .
10.6
+ . .
. .
Cytocentrifuged sperm were treated with the above detergents for 30 min and the effects on their subsequent reactivity with MoAbs determined by immunofluorescence. + , Antigenicity decreased; + , antigenicity partly decreased; - , antigenicity not decreased.
by this treatment. The Mabs did not show any reactions with mouse or rabbit sperm when tested by immunofluorescence. None of the antibodies produced by cell lines 10.1-10.8 showed obvious sperm agglutinating activity (see summary Table 3). When the Mabs were tested by panning they fell into two categories: those that caused attachment to the panning plates in very large numbers (see Fig. 2a) and those that did not produce these effects (Fig. 2b). The Mabs positive in this assay were the same ones that were positive in the agglutination assay (see s u m m a r y Table 3).
TABLE 2 Anti-sperm activities of MoAb supernatants and control sera assessed by ELISA assay Solutions
Optical densities (SE)
tested
' Unblocked' target sperm
Target s p e r m ' blocked' with 15% FCS
1.12 (0.13)
0.70 (0.12)
0.69 0.04 0.03 0.03 0.05 0.05 0.08 0.06 0.09 0.06 0.04 0.04
0.46 0.00 0.04 0.07 0.01 0.03 0.04 0.10 0.12 0.06 0.05 0.02
Mouse anti-human sperm serum, 1 / 4 0 Normal mouse serum, 1 / 4 0 M o A b 10.1 M o A b 10.2 M o A b 10.3 M o A b 10.4 M o A b 10.5 M o A b 10.6 M o A b 10.7 MoAb 10.8 MoAb 10.9 MoAb 10.10 M o A b 10.11
(0.04) (0.01) (0.01) (0.01) (0.00) (0.02) (0.01) (0.01) (0.03) (0.02) (0.01) (0.01)
(0.05) (0.00) (0.01) (0.01) (0.01) (0.01) (0.01) (0.04) (0.05) (0.01) (0.03) (0.01)
Assay carried out as described in text. All MoAb supernatants showed less anti-sperm activity than 1 / 4 0 dilution of normal mouse serum.
220
b Fig. 2. (a) Human sperm treated with MoAb 10.11 bound to 'panning' plate in large numbers ( × 285). (b) Human sperm treated with MoAb 10.2 showing no substantial binding to 'panning' plate ( × 285).
221
TABLE 3 S u m m a r y o f a n t i - s p e r m activities of M o A b s a s s a y e d b y d i f f e r e n t p r o c e d u r e s Assays
Immunofluorescence Agglutination ' Panning' ELISA
Monoclonal antibodies 10.1
10.2
10.3
10.4
10.5
10.6
10.7
10.8
10.9
10.10
10.11
+ . .
+
+
+ .
+
+
+
+
+ +
+ +
+ +
.
.
.
.
.
. .
. .
. .
. . .
. .
. .
.
.
.
A n t i b o d y class: M o A b s 1 0 . 1 - 1 0 . 8 , I g G l ; M o A b s 1 0 . 9 - 1 0 . 1 1 , I g M .
None of the Mabs was positive in the ELISA assay described. All showed less binding to the glutaraldehyde fixed sperm than did the immunoglobulin in the normal mouse serum negative control (Table 2). This was observed with both 'unblocked' target sperm and target sperm blocked with 15% FCS. The Mabs similarly failed to show anti-sperm activity when sperm treated with 0.5% glutaraldehyde (Witkin, 1983) were used as targets (data not shown). The antibody classes of the Mabs are given in Table 3.
Discussion
The results reported in this paper clearly demonstrated that different sperm-antibody interactions were detected with differing sensitivities by the different assays (see summary Table 3). The results reported have obvious important practical implications since they emphasize that one should be cautious in assuming that the anti-sperm activities detected in sera by different assays are in fact measuring the same interactions. This applies to all new procedures but probably particularly to the ELISA procedures because their apparently greater convenience (compared with sperm agglutination) may tempt inexperienced workers to use such procedures uncritically for the clinical assessment of anti-sperm immunity. The reasons for some of the differences reported are not obvious, but appear scientifically interesting and are under further investigation. It is not surprising that IgM antibodies should be more readily detectable by an agglutination assay than IgG, but the reasons for the failure to detect the anti-sperm activities of Mabs 10.9, 10.10 and 10.11 by immunofluorescence are less clear. One hypothesis would be that the antigens with which these Mabs react are present at a low density on the membrane, sufficient to allow agglutination but insufficient to produce detectable immunofluorescence. An alternative hypothesis would be that the molecules are lost from the sperm or lose their antigenic conformation when the sperm are cytocentrifuged and repeatedly washed. There is no reason to suppose that the failure to detect the activity was caused by inability of the FITC-conjugated anti-mouse immunoglobulin to bind to IgM since the anti-immunoglobulin was directed against both heavy and light chains and reacted with IgM conjugated to Sepharose beads. In
222 addition, a specific FITC-conjugated anti-IgM (a generous gift from R.M.E. Parkhouse) failed to show any convincing fluorescence with these Mabs on sperm. Discrepancies between the results of immunofluorescence and sperm agglutination assays have also been reported when these procedures were used to test human sera (e.g. Menge, 1980; Hancock, 1981). The observation that the Mabs which showed strong sperm-agglutinating activity also showed very striking positive reactions in the panning procedure while the Mabs negative in the agglutination assay did not (see Table 3) was also interesting. Whether this occurred because of differences between the antigens with which the different Mabs were reacting or solely because of differences in immunoglobulin class cannot be readily determined. There is, however, evidence that the panning assay is capable of detecting anti-sperm IgG (Hancock and Faruki, 1984) which would, to a degree, argue against the second hypothesis. One could speculate that the panning assay might be more sensitive for more superficial antigens (which would also be detected in agglutination assays) and be less sensitive for less superficial antigens which might become exposed, and therefore detected, by the immunofluorescence procedure. The observation that the antigens reacting with Mabs 10.1-10.8 were not removed by Triton treatment (Table 1) would be consistent with their being less superficial antigens (Jones et al., 1983). Although anti-sperm activity could be detected in mouse anti-human sperm serum (see Table 2) using the ELISA procedure described, this assay clearly did not demonstrate anti-sperm activity in the supernatants tested. In fact, the normal mouse serum negative controls showed substantially more anti-sperm activity than the Mab supernatants. This was not observed with any of the other assays used. The binding of immunoglobulin from normal mouse serum to sperm was increased when the sperm were treated with glutaraldehyde (data not shown - tested by immunofluorescence). Whether the binding of immunoglobulin from normal mouse serum to glutaraldehyde-treated sperm in the ELISA assay described reflects the binding of 'natural' anti-sperm antibodies (see Hancock, 1981, 1984 for recent reviews) or non-specific binding of immunoglobulin via the Fc part of the molecule (e.g. Witkin et al., 1980) was not determined. However, whatever the basis, the phenomenon is worth bearing in mind when considering the use of glutaraldehyde for treating target sperm. It clearly makes the sperm unsuitable for the assessment of some antigen-antibody interactions. In summary, we have found that different sperm-monoclonal antibody interactions were detected with differing sensitivities by different assays. These results have obvious practical implications and the biological reasons for them are under further investigation.
Acknowledgements We would like to thank our colleagues, particularly G. Preece and M. Kennedy, for their help and advice, D. Wraith for reading the manuscript and M. Bertagne for typing it. We thank W.F. Hendry, J. Stedronska and C. Stock for arranging for the supplies of human sperm from the Seminology Laboratory at Chelsea Hospital.
223 References Birkeland, S.A. (1980) Cryopreservation of human lymphocytes for sequential testing of immune competence. J. Immunol. Methods 35, 57-67. Fazekas de St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1-21. Friberg, J. (1974) Clinical and immunological studies on sperm-agglutinating antibodies in serum and seminal plasma. Acta Obstet. Gynaecol. Scand., Suppl. 36, 3-76. Hafez, E.S.E. (1970) Rabbits. In Reproduction and Breeding Techniques for Laboratory Animals (Hafez, E.S.E., ed.) pp. 273-298. Lea and Febiger, Philadelphia. Hancock, R.J.T. (1981) Immune responses to sperm. In Oxford Reviews of Reproductive Biology III (Finn, C.A., ed.), pp. 183-208. Clarendon Press, Oxford. Hancock, R.J.T. (1984) Immune responses to sperm - recent developments. In Proceedings of 38th Sutton Bonnington Easter School: Immunological Aspects of Reproduction in Mammals (Crighton, D.B., ed.), pp. 55-76. Butterworths, London. Hancock, R.J.T. and Faruki, S. (1984) Detection of antibody-coated sperm by 'panning' procedures. J. Immunol. Methods 66, 149-159. Hudson, L. and Hay, F.C. (1980) Practical Immunology, Second Edition. Blackwell Scientific Publications, Oxford, p. 315. Jones, R., Brown, C.R., Cran, D.G. and Gaunt, S.S. (1983) Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies. Gamete Res. 8, 255-265. Lee, C.G., Huang, Y., Huang, C., Hu, P.C. and Menge, A.C. (1982) Monoclonal antibodies to human sperm antigens. J. Reprod. Immunol. 4, 173-181. Menge, A.C. (1980) Clinical immunologic infertility: diagnostic measures, incidence of antisperm antibodies, fertility and mechanisms. In Immunological Aspects of Fertility Regulation (Dhindsa, D.S. and Schumacher, F.B., eds.), pp. 205-224. Elsevier/North-Holland, Amsterdam. Menge, A.C., Richter, D.E., Naz, R.K., Lee, C.G. and Wong, E. (1983) Dissecting the sperm cell by use of monoclonal antibodies. In Reproductive Immunology 1983 (Isojima, S. and Billington, W.D., eds.), pp. 81-90 Elsevier Science Publishers, Amsterdam. Richardson, D.W. (1976) Techniques of sperm storage. In Artificial Insemination: Proceedings of the 4th Study Group of the Royal College of Obstetricians and Gynaecologists (Brudenell, M., McLaren, A., Short, R.V. and Symonds, M., eds.), pp. 97-106. Royal College of Obstetricians and Gynaecologists, London. Witkin, S.S. (1983) Enzyme linked immunosorbent assay (ELISA) for detection of antibodies to spermatozoa. Res. Reprod. 15, 1. Witkin, S.S., Shahani, S.K., Gupta, S., Good, R.A. and Day, N.K. (1980) Demonstration of IgG Fc receptors on spermatozoa and their utilization for the detection of circulating complexes in human serum. Clin. Exp. Immunol. 41,441-452. Witkin, S.S., Zelikovsky, G., Good, R.A. and Day, N.K. (1981) Demonstration of llSIgA antibody to spermatozoa in human seminal fluid. Clin. Exp. Immunol. 44, 368-374. Wolf, D.P., Sokoloski, J.E., Dandekar, P. and Bechtol, K.B. (1983) Characterization of human sperm surface antigens with monoclonal antibodies. Biol. Reprod. 29, 713-723. Zanchetta, R., Busolo, F. and Mastrogiacomo, I. (1982) The enzyme-linked immunosorbent assay for detection of the anti-spermatozoal antibodies. Fertil. Steril. 38, 730-734.