title: Enhancement in antigen binding by a combination of synergy and antibody capture.
abstract: The effects of orientating pairs of synergistic monoclonal antibodies (mAb) on binding of human chorionic gonadotropin (hCG) was studied by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Antibody synergy towards hCG required two functionally intact antibodies located adjacent to each other and with different epitope specificities. We investigated whether immobilization procedures avoiding protein denaturation, increasing proper orientation and promoting higher molecular flexibility of the synergistic mAb resulted in significantly enhanced antigen, binding. Synergistic mAb pairs captured through their Fc-region by protein G or a polyclonal serum against the Fc-part of mouse IgG could be used at 10-fold lower coating concentrations to achieve maximal binding of the analyte as compared with the same mAb pairs coated directly onto polystyrene. The synergistic effect observed with protein A used as capture varied greatly with the subclasses of the two synergistic antibodies employed. Scatchard analysis revealed that the number of functionally synergistic antibody sites participating in the binding of hCG for one mAb pair was about 10 times higher for the protein G-captured as compared with the directly coated synergistic pair. Biotinylated synergistic mAb pairs, coated directly or captured by streptavidin, did not display any enhanced antigen binding when tested in SPR or ELISA. With SPR, synergy was only observed when the synergistic mAb had been captured through their Fc-region. Using protein G or a polyclonal rabbit anti-IgG1 serum as capture reagents in SPR, synergistic triple mAb combinations against hCG were demonstrated.
authors.affiliation: Department of Immunology, University College London Medical School, UK.
authors.forename: T
authors.initials: T
authors.lastname: Klonisch
authors.forename: G
authors.initials: G
authors.lastname: Panayotou
authors.forename: P
authors.initials: P
authors.lastname: Edwards
authors.forename: A M
authors.initials: AM
authors.lastname: Jackson
authors.forename: P
authors.initials: P
authors.lastname: Berger
authors.forename: P J
authors.initials: PJ
authors.lastname: Delves
authors.forename: T
authors.initials: T
authors.lastname: Lund
authors.forename: I M
authors.initials: IM
authors.lastname: Roitt
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D006063:Chorionic Gonadotropin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007158:Immunologic Techniques; D009419:Nerve Tissue Proteins / Q000276:immunology; D011863:Radioimmunoassay
chemical_list: D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D000939:Epitopes; C029152:G-substrate; D007141:Immunoglobulin Fc Fragments; D009419:Nerve Tissue Proteins