BISPECIFIC MULTIVALENT ANTIBODY STUDIED BY REAL-TIME INTERACTION ANALYSIS FOR THE DEVELOPMENT OF AN ANTIGEN-INHIBITION ENZYME-LINKED-IMMUNOSORBENT-ASSAY

A bispecific antibody with specificities for both 7-hydroxycoumarin (7 -OHC) and alkaline phosphatase (AP) was produced by chemically cross-l inking two parental polyclonal antibodies, Real-time interaction analy sis of the bispecific multivalent antibody (bsMAb) was performed using BIAcore, a surface plasmon resonance (SPR)-based biosensor, in order to confirm its bispecific nature, A 7-OHC-BSA conjugate was covalently immobilized to a dextran matrix to serve as the reaction surface and unconjugated bovine serum albumin (BSA) was immobilized on to a separa te dextran matrix as a control surface, Immunoaffinity-purified bsMAb, parental anti-7-OHC antibody and AP were injected over both surfaces, The bsMAb was shown to bind both antigens, 7-OHC and AP, simultaneous ly, Comparison of the ratio of mass bound for bsMAb and AP (5:1) with the ratio of the molecular masses of bsMAb (approximately 300 kDa) and AP (85 kDa) (3.5:1) suggests that most of the bsMAb species possess b oth specificities, The bsMAb was employed in a one-step antigen-inhibi tion ELISA for the detection of 7-OHC, The assay was compared with a c onventional ELISA approach employing an AP-labelled secondary antibody , The bispecific antibody approach proved to be faster and more sensit ive, with a detection limit of 6 ng ml(-1) as compared with approximat ely 50 ng ml(-1) for the conventional approach, The assay was used for the quantification of free and total 7-OHC in urine samples from two healthy volunteers who had been administered coumarin, The accuracy an d precision of the assay were assessed, The bispecific antibody-based assay gave similar results, accuracy and precision, but proved to be f ar more sensitive (limit of determination 6 ng ml(-1) for total 7-OHC) , It is concluded that real-time interaction analysis using BIAcor pro vides a rapid method for the evaluation of the bsMAb and it was verifi ed that the bispecific product formed by chemical cross-linking of two parental antibodies offers a simple alternative for the development o f a highly sensitive ELISA.