A new method for the determination of the ABO blood type of semen by immunoblotting using anti-ABH antibodies following immunoprecipitation

A new method that enables the ABO blood type of semen to be determined by identifying the molecular weight of a blood group substance (BGS) has been developed. In order to produce an antibody against a BGS (p84) detected on the sperm plasma membrand (SPM), p84 was purified from seminal plasma, injected into a rabbit. When seminal plasma was immunoprecipitated with the anti-p84 antibody, SDS-PAGE analysis of the immune complex yielded three specific polypeptides with molecular masses of 84, 51 and 25 kDa. The first of these polypeptides, p84, showed ABH antigenic activity when subjected to immunoblotting. The 51 and 25 kDa proteins corresponded to rabbit IgG heavy and light chains, respectively. Using this method, ABH antigenic activity of a band with a molecular mass of 84 kDa was detected in 25 seminal plasma samples by immunoblotting. When serial two-fold dilutions of seminal plasma were analyzed using this method, clear immunoreactive 84 kDa bands were observed up to 60-fold dilution. When seminal plasma was mixed with vaginal fluid at varying ratios, ABH antigenic activity of 84 kDa immunoreactive bands could be detected until the seminal plasma: vaginal fluid mixture reached a ratio of 1:20. These results suggest that this method will be applicable to the analysis of forensic samples of semen contaminated by vaginal fluid, such as those obtained from victims of sexual assault.