An enzyme-linked immunosorbent assay for measuring antisperm autoantibodies following vasectomy in Lewis rats

An indirect enzyme-linked immunosorbent assay (ELISA) was devised to measure antisperm auto-antibodies in the Lewis rat following vasectomy. The assay system was validated by employing prevasectomy sera and postvasectomy antisera, previously demonstrated to contain antisperm antibodies by indirect immunofluorescence. A standardized ELISA protocol was developed employing 10(5) sperm per microtiter plate well and sucrose-polyvinylpyrrolidone as a postcoat stabilizer solution. The ELISA was shown to yield significant detectable antibody at dilutions of 1/512 or greater in the most reactive sera. A standard for scoring positive titers was adopted: 1.96 standard deviations above the mean of the preimmune value. Using the criterion, 88% of 7-week postvasectomy samples could be discriminated from preimmune samples at a 1:16 dilution, which was adopted for subsequent assays. The ELISA demonstrated 73% and 91% reproducibility for an intraassay analysis of single prevasectomy and postvasectomy serum samples (7 weeks postvasectomy) tested in 160 determinations on a standard sperm pool. When this single antigen pool was employed in 35 determinations at 0, 1, and 4 weeks in an interassay study, 56% and 70% reproducibility was found for pre- and postvasectomy sera respectively. A correlation (r = 0.75) was made between a single absorbance value and the endpoint titer of the same sera, which indicated that single absorbance values could be used to predict serum titer and single dilutions could be used for general screening of a large number of samples. The ELISA described provides a rapid, sensitive, and reliable method that discriminated between samples taken before and after vasectomy.