An enzyme immunoassay for the quantitative determination of antibodies directed against spermatozoa antigens in serum. The Sperm Antibody ELISA (Enzyme Linked ImmunoSorbent Assay) is a solid-phase sandwich enzyme-immunoassay for the quantitative determination of anti-spermatozoa antibodies in human serum. The ELISA-plate is coated with a mix of spermatozoa proteins which are recognized by anti-spermatozoa antibodies. The samples and standards are pipetted into the wells and then incubated. During this incubation anti-spermatozoa antibodies bind to the spermatozoa proteins and are thus immobilised on the plate. After washing the enzyme conjugate, consisting of anti-human globulin antibodies covalently coupled to horseradish peroxidase, is added. After removal of the unbound conjugate by washing the horseradish peroxidase oxidizes the then added substrate TMB (3,3',5,5'-tetramethylbenzidine) yielding a color reaction which is stopped with 0.25 M sulphuric acid (H2SO4). The extinction is measured at a wavelength of 450 nm with a microplate reader. The use of a reference measurement with a wavelength ≥550 nm is recommended.