An Enzyme-Linked Immunosorbent Assay (ELISA) for Human Semen Identification Based on a Biotinylated Monoclonal Antibody to a Seminal Vesicle-Specific Antigen

Monoclonal antibody mouse antihuman semen-5 (MHS-5) (immunoglobulin G1 [IgG1]) was biotinylated using N-biotinyl-w-aminocaproic acid-N-hydroxysuccinimide ester. This monoclonal antibody-biotin conjugate recognized low molecular weight peptide bands between 10.5 and 20 kilodaltons on immunoblots of liquefied semen. Immunodominant peptides had molecular weights of 10.5, 11.5, and 13.5 kilodaltons. An enzyme-linked immunosorbent assay (ELISA) developed with the biotinylated-MAb and streptavidin peroxidase demonstrated sensitivity curves with lower limits of 10 ng of seminal fluid protein per microtiter well using 50 ng per well of monoclonal antibody-biotin conjugate. Cross-reactivity studies on a panel of human biological fluids and tissues demonstrated no cross-reactivity or false positives using the monoclonal antibody-biotin conjugate. The sensitivity of the monoclonal antibody-biotin ELISA was compared to ELISA based upon a polyclonal secondary antibody-peroxidase conjugate. These findings indicate that this ELISA assay, based on a biotinylated monoclonal antibody to a seminal vesicle-specific antigen, may be useful for semen identification.