Synergistic effects in antigen capture ELISA using three monoclonal antibodies directed at different epitopes of the same antigen

• Published in: Clinica chimica acta 2000-09, Vol.299 (1), p.25-44
• Main Authors: Nikulina, Valeria A, Kizim, Elena A, Massino, Yulia S, Segal, Olga L, Smirnova, Maria B, Avilov, Vladimir V, Saprigin, Dmitri B, Smotrov, Sergei P, Tichtchenko, Valentina A, Kolyaskina, Galina I, Dmitriev, Alexander D
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Antibody Specificity; Antigen capture ELISA; Antigens - chemistry; Calibration; Cells, Cultured; Chromatography, Affinity; Chromatography, Ion Exchange; Enzyme-Linked Immunosorbent Assay - methods; Epitopes - chemistry; Horseradish Peroxidase - chemistry; Human myoglobin; Humans; Mice; Mice, Inbred BALB C; Monoclonal antibodies; Myocardium - chemistry; Myoglobin - immunology; Proteins - chemistry; Rabbits; Reference Standards; Synergistic effect; Three-site
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/S0009-8981(00)00269-2

Using a panel of monoclonal antibodies (mAb) against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture enzyme-linked immunosorbent assay (ELISA) may be significantly increased by the simultaneous immobilization on a solid phase of two co-operating capture mAbs. This method (“a three-site ELISA”) uses three mAbs at different epitopes of the same antigen (two capture/one tracer), unlike the traditional two-site assay, using one capture and one tracer mAbs. We established two-site and three-site ELISA assays for Mb, by varying capture and tracer mAbs. Three-site assays showed 4–6 fold increase in sensitivity, if compared with two-site assays. The model for the effect has been suggested, according to which in three-site ELISA the high-affinity cyclic configurations may be formed by an antigen, two-capture mAbs and the surface of solid phase.