Klonisch T, Panayotou G, Edwards P, Jackson A M, Berger P, Delves P J, Lund T, Roitt I M
Department of Immunology, University College London Medical School, UK.
Immunology. 1996 Oct;89(2):165-71. doi: 10.1046/j.1365-2567.1996.d01-722.x.
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.
通过放射免疫测定法（RIA）、酶联免疫吸附测定法（ELISA）和表面等离子体共振（SPR）研究了定向协同单克隆抗体（mAb）对人绒毛膜促性腺激素（hCG）结合的影响。抗体对hCG的协同作用需要两个功能完整且彼此相邻且具有不同表位特异性的抗体。我们研究了避免蛋白质变性、增加正确定向并促进协同mAb更高分子灵活性的固定程序是否会导致抗原结合显著增强。与直接包被在聚苯乙烯上的相同mAb对相比，通过蛋白G或针对小鼠IgG Fc部分的多克隆血清通过其Fc区域捕获的协同mAb对可在低10倍的包被浓度下使用，以实现分析物的最大结合。用作捕获剂的蛋白A所观察到的协同效应因所使用的两种协同抗体的亚类而异。Scatchard分析表明，与直接包被的协同对相比，对于一个mAb对，参与hCG结合的功能协同抗体位点数量对于蛋白G捕获的大约高10倍。直接包被或被链霉亲和素捕获的生物素化协同mAb对在SPR或ELISA测试中未显示任何增强的抗原结合。使用SPR时，仅当协同mAb通过其Fc区域被捕获时才观察到协同作用。在SPR中使用蛋白G或多克隆兔抗IgG1血清作为捕获试剂，证明了针对hCG的协同三联mAb组合。
