121
Journal of lmmunological Methods, 142 (1991) 121-126 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100268P JIM 06038
Evaluation of a solid-phase cellular enzyme immunoassay for detection of the serologically defined male antigen Brian D. Reilly * and Ellen H. Goldberg Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland A1B 31/6, Canada, and Department of Microbiology, School of Medicine, Unicersity of New Mexico, Albuquerque, NM, U.S.A. (Received 15 November 1990, revised received 8 May 1991, accepted 8 May 1991)
An indirect cellular enzyme immunoassay for the detection of the serologically defined male-specific antigen, SDMA, was developed using mouse spermatozoa as the target. Serum from B6 female mice injected with male spleen cells was used as the source of SDMA-specific antibody. Our results indicate that the assay is highly reliable (96% accurate) with an intra- and interassay coefficient of variation < 12%. Since this assay is non-subjective and simple to perform it provides a useful alternative to the complement-dependent cytotoxicity assay for detection of SDMA. Key words: H-Y antigen; Serologically defined male-specific antigen; Enzyme immunoassay
Introduction
H-Y was first described as a cell-surface antigen responsible for the rejection of male skin grafts by otherwise histocompatible females of the same inbred mouse strain (Eichwald and Silmser, 1955). With the serological detection of H-Y on mouse spermatozoa using serum from female mice which had rejected multiple male skin grafts (Goldberg et al. 1971), it became possible to study the phylogenetic distribution of the antigen. It was found that H-Y was highly conserved throughout evolution of the vertebrate resulting in the hypothesis that H-Y plays a primary role in male sex determination (Wachtel et al.,
Correspondence to: E.H. Goldberg, Department of Microbiology, School of Medicine, University of New Mexico, Albuquerque, NM87131, U.S.A. * B.D. Reilly is the recipient of a N.I.H. Postdoctoral Fellowship.
1975). In addition, it has been suggested that the antigen detected serologically is different than H-Y, defined by transplantation, leading to the designation of SDMA (serologically-defined male antigen; Silvers et al., 1982). It became clear, with numerous laboratories using a variety of detection methods and different sources of malespecific antisera, that detection of SDMA in individual mice was not always reproducible. This led to a situation which made comparison of independently derived experimental results difficult (Goodfellow and Andrews, 1982). Consequently, the validity of serological assays for the detection of SDMA has been challenged by the fact that difficulties have been encountered in obtaining consistent results which are reproducible in different laboratories. Since analysis of SDMA is dependent on serological determination, reproducibility and consistency of the assay is critical. The standard serological assay for SDMA has been the complement-dependent cytotoxicity assay using spermatozoa as the target (Goldberg et
122 al., 1971). This assay is technically difficult to perform, and if uncoded samples are used the results are subject to personal interpretation. Recently, a study describing the development of an enzyme-linked immunosorbant assay (ELISA) as an alternative method of detection of SDMA was reported (Farber et al., 1984; Brunner and Wachtel, 1988). This assay used testis supernatant, apparantly containing the soluble male antigen, as the target. Since soluble SDMA may be different from the membrane-bound form we chose to evaluate the applicability of a cellular enzyme immunoassay (CELISA) for SDMA detection.
Materials and methods
Preparation of antiserum Polyclonal male-specific antiserum was produced in C57BL/6 (B6) female mice by inoculation with 25 x 106 B6 male spleen cells every 2 weeks. Animals were individually bled via the tail vein on days 7, 10 and 14 after the fifth and subsequent inoculations. Serum was collected and tested for antibody against SDMA by the conventional complement-dependent cytotoxicity assay (Goldberg et al., 1971). Serum which was known to contain SDMA-specific antibody was pooled and stored at - 7 0 °C.
Preparation of spermatozoa Sexually mature C57BL/6 male mice were killed by cervical dislocation and epididymides were placed in phosphate-buffered saline (PBS) at 37°C, 5% CO 2. The PBS contained 5% 3'globulin-free, heat-inactivated fetal calf serum (IPT) with 0.001 M CaCI 2 and 0.1% fructose. The caudae epididymis were opened by making several small incisions, sperm were allowed to disperse into the medium for 20-30 min and then gently pipetted through a 110 /xm nylon mesh into a plastic Falcon centrifuge tube. An aliquot was counted in a hemocytometer and the concentration adjusted to 2 x 106 cells/ml.
Absorption analysis Details of the absorption procedure are described by Boyse et al. (1970). Essentially, SDMA specific antiserum, at a one in four dilution, un-
less indicated otherwise, was incubated with B6 male splenocytes (1 volume packed cells: 4 volume serum) for 30 min in an ice-bath. After removing the ceils by centrifugation, the serum was tested for the presence of SDMA antibody against sperm by the sperm cytotoxicity assay as described (Goldberg et al., 1971). To control for specificity, antiserum was absorbed with B6 female spleen cells.
Cellular enzyme-linked immunosorbant assay (CELISA) 50 /zl of 0.1 m g/ m l poly-L-lysine (molecular weight 60-100 kDa) was added to each well of a 96-well, flat-bottomed, polystyrene microtitration plate and allowed to air-dry. After thorough washing with PBS, 100 /zl of sperm suspension (2 X 106 cells/ml) was added to each well. The plate was centrifuged at 300 x g for 5 min followed by the addition of 25 /xl of 2.5% paraformaldehyde (E.M. grade) in PBS containing 5% dextrose, pH 7.2. The cells were fixed for 30 rain. After four washes with PBS the wells were filled with 1% 3,-globulin-free fetal calf serum (IPT) in PBS overnight at 4°C to block potential sites of non-specific binding. 50 tzl of serially diluted unabsorbed serum or serum previously absorbed with spleen cells from B6 male or female mice was added to appropriate wells and allowed to incubate for 2.5 h at 4°C followed by four washes with PBS. A 100 /zl sample of horseradish peroxidase labeled goat anti-mouse kappa chain antibody (Southern Biotechnology Associates, Inc.; diluted 1 in 4000 in PBS with 1% IPT) was added and incubated for 90 min at 4°C. After four washes with PBS 100 /xl of the substrate containing 2,2-azino-di-3-ethyl benzthiazoline-sulfonic acid (ABTS) and peroxide in a citric acid buffer, pH 4.0 was added. Following an incubation period of 30 min at room temperature the absorbance of each well was determined by reading the plate at 405 nm in a Biotek ELISA plate reader. All tests were performed in triplicate unless otherwise indicated. The background absorbance was obtained from wells in which only the horseradish peroxidase-labeled goat antimouse serum was added followed by the ABTS. Statistical significance between the activity of serum samples absorbed with female as com-
123
pared to male spleen cells was determined by Student's T-distribution. Those samples with a P value < 0.05 were considered positive for SDMA antibody.
Results
To evaluate the accuracy of CELISA for SDMA detection, sera from 9 B6 females immunized against male cells and 8 females immunized against female cells were tested. Results presented in Table I show a significant difference in activity of serum from B6 females immunized with male spleen cells after absorption with male cells when compared to activity present in the serum after absorption with female cells (see Group A). In contrast, there was essentially no difference in activity of sera from females immunized with female cells after absorption with male
or female spleen cells (Table I, Group B) indicating absence of SDMA antibody. Of the 17 samples tested by CELISA, serum from only one female immunized with male cells lacked antibody to SDMA. In no case did mice immunized with female cells produce SDMA-specific antibody. These results indicate a high degree of reliability. Data presented in Fig. 1 show the average reaction of the nine samples from Table I, group A titrated in the CELISA. We found a significant difference (P < 0.01) in binding to sperm cells at a 1/4, 1 / 8 and 1/16 dilution of serum absorbed with male cells compared to unabsorbed serum or serum absorbed with female cells. Additionally, we were unable to find any significant difference in sperm binding by the male absorbed serum when compared to the serum from mice prior to immunization with male cells (i.e. preimmune serum).
TABLE I M E A S U R E M E N T OF T H E P R E S E N C E O F SDMA A N T I B O D Y U S I N G T H E ELISA A G A I N S T SPERM T A R G E T CELLS Group
Immunization
Sera tested
Serum absorbed with 9 cells a
Serum absorbed with d cells a
Difference in the 9 absorbed minus the d absorbed serum
p b -value
A
B6 female mice inoculated with B6 male spleen cells
1 2 3 4 5 6 7 8 9
0.85 ± 0.03 0.49 5:0.05 0.42 ± 0.03 0.38 ± 0.04 0.51 ± 0.08 0.70 ± 0.04 0.51 ±0.01 0.27 + 0.02 0.96 ± 0.01
0.73 + 0.01 0.38 + 0.01 0.29 + 0.03 0.26 + 0.03 0.38 + 0.01 0.59 + 0.01 0.36-t-0.01 0.28 + 0.01 0.79 + 0.02
0.12 0.11 0.13 0.12 0.13 0.11 0.15 0.00 0.17
< 0.05 < 0.05 < 0.05 < 0.05 < 0.01 < 0.01 < 0.05 NS < 0.05
B
B6 female mice inoculated with B6 female spleen cells
1 2 3 4 5 6 7 8
0.80 ± 0.02 0.69 ± 0.04 0.40 ± 0.04 0.38 ___0.06 0.86 5- 0.01 0.68 ± 0.05 0.93 ± 0.01 0.44 5:0.04
0.76 + 0.07 0.67 5:0.03 0.38 + 0.01 0.33 5:0.02 0.84 + 0.02 0.59 5:0.04 0.95 ± 0.03 0.45 + 0.02
0.04 0.01 0.02 0.05 0.02 0.09 0.00 0.00
NS NS NS NS NS NS NS NS
a Prior to testing, sera were absorbed with equivalent numbers of either male or female spleen cells as described in materials and methods. Unabsorbed and absorbed sera were titrated in the CELISA (see Fig. 1). The results presented are the mean absorbance values obtained in the CELISA using serum at a 1 / 1 6 dilution + standard deviation. All sera were run in triplicate. b Significance of the difference between serum absorbed with female cells vs. the same serum absorbed with male cells as determined by Student's t-test. NS = not significant.
124 08
Precision of the assay
-UNABSORBED SERUM ABSORBED WITH FEMALE CELLS
The precision of the assay was tested by two methods. First intra-assay variation was determined by testing three individual SDMA-specific antisera (previously absorbed with equivalent numbers of either male or female spleen cells) 6 times in one assay. Second, interassay variation was determined by assaying the three serum samples in triplicate in 5 consecutive assays. The results presented in Table II indicate that the intra-assay coefficient of variation was 9% or less while the interassay precision was 12% or less.
0.7
06
0.5
0.4
0.3
Reproducibility of the CELISA Reproducibility of the assay was determined using a known SDMA-specific antiserum at a 1 / 1 6 dilution. On day zero six microtitre plates lO
20
30
40
RECIPROCAL DILUTION o f t h e ANTISERUM
Fig. 1. Detection of S D M A antibody using serum from B6 female mice immunized with syngeneic male spleen cells. Serum was absorbed with equivalent n u m b e r s of either male (11) or female ( x ) spleen cells and titrated against B6 epididymal sperm in the C E L I S A as described in materials and methods. Results are compared to unabsorbed serum ( • ) and serum obtained before immunization ( • ) . The data are the m e a n of 9 samples _+standard deviation. Each sample was done in triplicate.
o UNABSORBED SERUM m SERUM BEFORE IMMUNIZATION = ABSORBED WITH FEMALE CELLS • ABSORBED WITH MALE CELLS 0.60
0.55
E
t-'-
u~
The differences presented in Fig. 1, between antiserum absorbed with male or female cells, while significant, is small. To confirm that our assay is specific for SDMA serial dilutions of both the antiserum and cells for absorption were incubated in a chessboard fashion. The results are presented in Fig. 2. For simplicity, the data show the average reaction of the 9 samples from Table I, group A at a 1 / 1 6 dilution. A similar pattern of reactivity was observed at a 1/4, 1/8, and a 1 / 3 2 dilution. The results show that absorption of SDMA-specific antiserum with increasing numbers of SDMA-specific spleen ceils has no effect on its binding activity to sperm cells. In contrast, absorption of SDMA specific-antiserum with increasing numbers of male spleen cells resulted in a steady decrease in binding activity to sperm. These results indicate the differences which we detect are specific and not quantitative.
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Journal of lmmunological Methods, 142 (1991) 121-126 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100268P JIM 06038
Evaluation of a solid-phase cellular enzyme immunoassay for detection of the serologically defined male antigen Brian D. Reilly * and Ellen H. Goldberg Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland A1B 31/6, Canada, and Department of Microbiology, School of Medicine, Unicersity of New Mexico, Albuquerque, NM, U.S.A. (Received 15 November 1990, revised received 8 May 1991, accepted 8 May 1991)
An indirect cellular enzyme immunoassay for the detection of the serologically defined male-specific antigen, SDMA, was developed using mouse spermatozoa as the target. Serum from B6 female mice injected with male spleen cells was used as the source of SDMA-specific antibody. Our results indicate that the assay is highly reliable (96% accurate) with an intra- and interassay coefficient of variation < 12%. Since this assay is non-subjective and simple to perform it provides a useful alternative to the complement-dependent cytotoxicity assay for detection of SDMA. Key words: H-Y antigen; Serologically defined male-specific antigen; Enzyme immunoassay
Introduction
H-Y was first described as a cell-surface antigen responsible for the rejection of male skin grafts by otherwise histocompatible females of the same inbred mouse strain (Eichwald and Silmser, 1955). With the serological detection of H-Y on mouse spermatozoa using serum from female mice which had rejected multiple male skin grafts (Goldberg et al. 1971), it became possible to study the phylogenetic distribution of the antigen. It was found that H-Y was highly conserved throughout evolution of the vertebrate resulting in the hypothesis that H-Y plays a primary role in male sex determination (Wachtel et al.,
Correspondence to: E.H. Goldberg, Department of Microbiology, School of Medicine, University of New Mexico, Albuquerque, NM87131, U.S.A. * B.D. Reilly is the recipient of a N.I.H. Postdoctoral Fellowship.
1975). In addition, it has been suggested that the antigen detected serologically is different than H-Y, defined by transplantation, leading to the designation of SDMA (serologically-defined male antigen; Silvers et al., 1982). It became clear, with numerous laboratories using a variety of detection methods and different sources of malespecific antisera, that detection of SDMA in individual mice was not always reproducible. This led to a situation which made comparison of independently derived experimental results difficult (Goodfellow and Andrews, 1982). Consequently, the validity of serological assays for the detection of SDMA has been challenged by the fact that difficulties have been encountered in obtaining consistent results which are reproducible in different laboratories. Since analysis of SDMA is dependent on serological determination, reproducibility and consistency of the assay is critical. The standard serological assay for SDMA has been the complement-dependent cytotoxicity assay using spermatozoa as the target (Goldberg et
122 al., 1971). This assay is technically difficult to perform, and if uncoded samples are used the results are subject to personal interpretation. Recently, a study describing the development of an enzyme-linked immunosorbant assay (ELISA) as an alternative method of detection of SDMA was reported (Farber et al., 1984; Brunner and Wachtel, 1988). This assay used testis supernatant, apparantly containing the soluble male antigen, as the target. Since soluble SDMA may be different from the membrane-bound form we chose to evaluate the applicability of a cellular enzyme immunoassay (CELISA) for SDMA detection.
Materials and methods
Preparation of antiserum Polyclonal male-specific antiserum was produced in C57BL/6 (B6) female mice by inoculation with 25 x 106 B6 male spleen cells every 2 weeks. Animals were individually bled via the tail vein on days 7, 10 and 14 after the fifth and subsequent inoculations. Serum was collected and tested for antibody against SDMA by the conventional complement-dependent cytotoxicity assay (Goldberg et al., 1971). Serum which was known to contain SDMA-specific antibody was pooled and stored at - 7 0 °C.
Preparation of spermatozoa Sexually mature C57BL/6 male mice were killed by cervical dislocation and epididymides were placed in phosphate-buffered saline (PBS) at 37°C, 5% CO 2. The PBS contained 5% 3'globulin-free, heat-inactivated fetal calf serum (IPT) with 0.001 M CaCI 2 and 0.1% fructose. The caudae epididymis were opened by making several small incisions, sperm were allowed to disperse into the medium for 20-30 min and then gently pipetted through a 110 /xm nylon mesh into a plastic Falcon centrifuge tube. An aliquot was counted in a hemocytometer and the concentration adjusted to 2 x 106 cells/ml.
Absorption analysis Details of the absorption procedure are described by Boyse et al. (1970). Essentially, SDMA specific antiserum, at a one in four dilution, un-
less indicated otherwise, was incubated with B6 male splenocytes (1 volume packed cells: 4 volume serum) for 30 min in an ice-bath. After removing the ceils by centrifugation, the serum was tested for the presence of SDMA antibody against sperm by the sperm cytotoxicity assay as described (Goldberg et al., 1971). To control for specificity, antiserum was absorbed with B6 female spleen cells.
Cellular enzyme-linked immunosorbant assay (CELISA) 50 /zl of 0.1 m g/ m l poly-L-lysine (molecular weight 60-100 kDa) was added to each well of a 96-well, flat-bottomed, polystyrene microtitration plate and allowed to air-dry. After thorough washing with PBS, 100 /zl of sperm suspension (2 X 106 cells/ml) was added to each well. The plate was centrifuged at 300 x g for 5 min followed by the addition of 25 /xl of 2.5% paraformaldehyde (E.M. grade) in PBS containing 5% dextrose, pH 7.2. The cells were fixed for 30 rain. After four washes with PBS the wells were filled with 1% 3,-globulin-free fetal calf serum (IPT) in PBS overnight at 4°C to block potential sites of non-specific binding. 50 tzl of serially diluted unabsorbed serum or serum previously absorbed with spleen cells from B6 male or female mice was added to appropriate wells and allowed to incubate for 2.5 h at 4°C followed by four washes with PBS. A 100 /zl sample of horseradish peroxidase labeled goat anti-mouse kappa chain antibody (Southern Biotechnology Associates, Inc.; diluted 1 in 4000 in PBS with 1% IPT) was added and incubated for 90 min at 4°C. After four washes with PBS 100 /xl of the substrate containing 2,2-azino-di-3-ethyl benzthiazoline-sulfonic acid (ABTS) and peroxide in a citric acid buffer, pH 4.0 was added. Following an incubation period of 30 min at room temperature the absorbance of each well was determined by reading the plate at 405 nm in a Biotek ELISA plate reader. All tests were performed in triplicate unless otherwise indicated. The background absorbance was obtained from wells in which only the horseradish peroxidase-labeled goat antimouse serum was added followed by the ABTS. Statistical significance between the activity of serum samples absorbed with female as com-
123
pared to male spleen cells was determined by Student's T-distribution. Those samples with a P value < 0.05 were considered positive for SDMA antibody.
Results
To evaluate the accuracy of CELISA for SDMA detection, sera from 9 B6 females immunized against male cells and 8 females immunized against female cells were tested. Results presented in Table I show a significant difference in activity of serum from B6 females immunized with male spleen cells after absorption with male cells when compared to activity present in the serum after absorption with female cells (see Group A). In contrast, there was essentially no difference in activity of sera from females immunized with female cells after absorption with male
or female spleen cells (Table I, Group B) indicating absence of SDMA antibody. Of the 17 samples tested by CELISA, serum from only one female immunized with male cells lacked antibody to SDMA. In no case did mice immunized with female cells produce SDMA-specific antibody. These results indicate a high degree of reliability. Data presented in Fig. 1 show the average reaction of the nine samples from Table I, group A titrated in the CELISA. We found a significant difference (P < 0.01) in binding to sperm cells at a 1/4, 1 / 8 and 1/16 dilution of serum absorbed with male cells compared to unabsorbed serum or serum absorbed with female cells. Additionally, we were unable to find any significant difference in sperm binding by the male absorbed serum when compared to the serum from mice prior to immunization with male cells (i.e. preimmune serum).
TABLE I M E A S U R E M E N T OF T H E P R E S E N C E O F SDMA A N T I B O D Y U S I N G T H E ELISA A G A I N S T SPERM T A R G E T CELLS Group
Immunization
Sera tested
Serum absorbed with 9 cells a
Serum absorbed with d cells a
Difference in the 9 absorbed minus the d absorbed serum
p b -value
A
B6 female mice inoculated with B6 male spleen cells
1 2 3 4 5 6 7 8 9
0.85 ± 0.03 0.49 5:0.05 0.42 ± 0.03 0.38 ± 0.04 0.51 ± 0.08 0.70 ± 0.04 0.51 ±0.01 0.27 + 0.02 0.96 ± 0.01
0.73 + 0.01 0.38 + 0.01 0.29 + 0.03 0.26 + 0.03 0.38 + 0.01 0.59 + 0.01 0.36-t-0.01 0.28 + 0.01 0.79 + 0.02
0.12 0.11 0.13 0.12 0.13 0.11 0.15 0.00 0.17
< 0.05 < 0.05 < 0.05 < 0.05 < 0.01 < 0.01 < 0.05 NS < 0.05
B
B6 female mice inoculated with B6 female spleen cells
1 2 3 4 5 6 7 8
0.80 ± 0.02 0.69 ± 0.04 0.40 ± 0.04 0.38 ___0.06 0.86 5- 0.01 0.68 ± 0.05 0.93 ± 0.01 0.44 5:0.04
0.76 + 0.07 0.67 5:0.03 0.38 + 0.01 0.33 5:0.02 0.84 + 0.02 0.59 5:0.04 0.95 ± 0.03 0.45 + 0.02
0.04 0.01 0.02 0.05 0.02 0.09 0.00 0.00
NS NS NS NS NS NS NS NS
a Prior to testing, sera were absorbed with equivalent numbers of either male or female spleen cells as described in materials and methods. Unabsorbed and absorbed sera were titrated in the CELISA (see Fig. 1). The results presented are the mean absorbance values obtained in the CELISA using serum at a 1 / 1 6 dilution + standard deviation. All sera were run in triplicate. b Significance of the difference between serum absorbed with female cells vs. the same serum absorbed with male cells as determined by Student's t-test. NS = not significant.
124 08
Precision of the assay
-UNABSORBED SERUM ABSORBED WITH FEMALE CELLS
The precision of the assay was tested by two methods. First intra-assay variation was determined by testing three individual SDMA-specific antisera (previously absorbed with equivalent numbers of either male or female spleen cells) 6 times in one assay. Second, interassay variation was determined by assaying the three serum samples in triplicate in 5 consecutive assays. The results presented in Table II indicate that the intra-assay coefficient of variation was 9% or less while the interassay precision was 12% or less.
0.7
06
0.5
0.4
0.3
Reproducibility of the CELISA Reproducibility of the assay was determined using a known SDMA-specific antiserum at a 1 / 1 6 dilution. On day zero six microtitre plates lO
20
30
40
RECIPROCAL DILUTION o f t h e ANTISERUM
Fig. 1. Detection of S D M A antibody using serum from B6 female mice immunized with syngeneic male spleen cells. Serum was absorbed with equivalent n u m b e r s of either male (11) or female ( x ) spleen cells and titrated against B6 epididymal sperm in the C E L I S A as described in materials and methods. Results are compared to unabsorbed serum ( • ) and serum obtained before immunization ( • ) . The data are the m e a n of 9 samples _+standard deviation. Each sample was done in triplicate.
o UNABSORBED SERUM m SERUM BEFORE IMMUNIZATION = ABSORBED WITH FEMALE CELLS • ABSORBED WITH MALE CELLS 0.60
0.55
E
t-'-
u~
The differences presented in Fig. 1, between antiserum absorbed with male or female cells, while significant, is small. To confirm that our assay is specific for SDMA serial dilutions of both the antiserum and cells for absorption were incubated in a chessboard fashion. The results are presented in Fig. 2. For simplicity, the data show the average reaction of the 9 samples from Table I, group A at a 1 / 1 6 dilution. A similar pattern of reactivity was observed at a 1/4, 1/8, and a 1 / 3 2 dilution. The results show that absorption of SDMA-specific antiserum with increasing numbers of SDMA-specific spleen ceils has no effect on its binding activity to sperm cells. In contrast, absorption of SDMA specific-antiserum with increasing numbers of male spleen cells resulted in a steady decrease in binding activity to sperm. These results indicate the differences which we detect are specific and not quantitative.
u.I 0 Z < rn or"
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