A UNIQUE AND SENSITIVE ELISA TECHNIQUE FOR TYPING ABH ANTIGENS IN BLOODSTAINS USING UEA-I LECTIN - THE REMOVAL OF DETERGENT WITH A SEPHADEXG-25 MINICOLUMN IMPROVES SENSITIVITY

A unique sandwich enzyme-linked immunosorbent assay (ELISA) for the de termination of ABH antigens in bloodstains has been developed. Human a nti-A and -B antisera and Ulex europaeus anti-H lectin were coated on the inner surface of microplate wells. The sample antigens from bloods tains, solubilized with n-octyl-beta-D-glucopyranoside which was then removed by passing through a Sephadex G-25 (G-25) mini-column, were pl aced in the wells. After washing the wells repeatedly, peroxidase-conj ugated Ulex europaeus lectin I was added and incubated. Antigen activi ties were determined by the development of colors using o-phenylenedia mine/H2O2. This technique permitted clear detection of all ABH antigen s corresponding to the antisera and lectin with high sensitivities. Th e A and B antigens were solubilized as aggregates with H antigen from the erythrocyte membrane. Excess detergent remaining in the sample red uced the sensitivity and accuracy of this ELISA, probably due to the r emoval of antibody from the wells by the effect of the surfactant. The treatment of solubilized antigens with G-25, an indispensable step, e liminated the adverse effect of the detergent on the ELISA. The ELISA method reported here was proved to be easy, economical and sensitive, and this technique should be useful in the forensic practice.