Substrate hydrolysis yields a signal that is inversely proportional to antigen concentration within the sample. A reporter-labeled secondary antibody is then added followed by the addition of substrate. The wells are washed to remove unbound antigen-antibody complexes and free antigen. These antibody-antigen complexes are then added to microtiter plates whose wells have been coated with purified antigen. The antigens in solution are in the test sample and are first incubated with antigen-specific antibody. The indirect competitive ELISA ( indirect c ELISA) pits plate-immobilized antigen against antigens in solution for binding to antigen-specific antibody.
Indirect Competitive Enzyme-Linked Immunosorbent Assay ( ELISA). However, there was evidence of autospecificity in a small percentage of males who had elevated levels of antisperm antibody by the direct assay that was not detected by the indirect assay using pooled donor spermatozoa. There was no statistically significant difference between results from the direct and indirect methods of the patients as a group. A simple linear regression analysis between the indirect and direct measurements of antisperm antibody resulted in a correlation coefficient of r = 0.907. The direct correlation between the results of these assays was 94%. Of the 66 infertile males, 21% (14/66) had elevated levels of antisperm antibody in their seminal plasma and 26% (17/66) had elevated levels bound directly to their spermatozoa. The normal concentration of sperm binding immunoglobulin was less than or equal to 1.5 fg Ig per spermatozoon for the indirect seminal plasma assay and less than or equal to 1.5 fg Ig per spermatozoon by the direct assay.
Comparison of a direct and indirect ELISA for quantitating antisperm antibody in semen.Ī direct and an indirect quantitative ELISA for antisperm antibody were compared using the spermatozoa and cell-free seminal fluid of 66 infertile males.
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