title: Bispecific multivalent antibody studied by real-time interaction analysis for the development of an antigen-inhibition enzyme-linked immunosorbent assay.
abstract: A bispecific antibody with specificities for both 7-hydroxycoumarin (7-OHC) and alkaline phosphatase (AP) was produced by chemically cross-linking two parental polyclonal antibodies. Real-time interaction analysis 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 separate 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, simultaneously. 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 both specificities. The bsMAb was employed in a one-step antigen-inhibition ELISA for the detection of 7-OHC. The assay was compared with a conventional ELISA approach employing an AP-labelled secondary antibody. The bispecific antibody approach proved to be faster and more sensitive, with a detection limit of 6 ng ml-1 as compared with approximately 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 and precision of the assay were assessed. The bispecific antibody-based assay gave similar results, accuracy and precision, but proved to be far more sensitive (limit of determination 6 ng ml-1 for total 7-OHC). It is concluded that real-time interaction analysis using BIAcor provides a rapid method for the evaluation of the bsMAb and it was verified that the bispecific product formed by chemical cross-linking of two parental antibodies offers a simple alternative for the development of a highly sensitive ELISA.
authors.affiliation: Institute of Immunology, Universität Witten/Herdecke, Germany.
authors.forename: H W
authors.initials: HW
authors.lastname: Reinartz
authors.forename: J G
authors.initials: JG
authors.lastname: Quinn
authors.forename: K
authors.initials: K
authors.lastname: Zänker
authors.forename: R
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis*; D018033:Antibodies, Bispecific / Q000032:analysis* / Q000302:isolation & purification; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D014468:Umbelliferones / Q000032:analysis*
chemical_list: D018033:Antibodies, Bispecific; D014468:Umbelliferones; C031477:7-hydroxycoumarin; D000469:Alkaline Phosphatase