MacKenzie C R, Hirama T, Deng S J, Bundle D R, Narang S A, Young N M
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
J Biol Chem. 1996 Jan 19;271(3):1527-33. doi: 10.1074/jbc.271.3.1527.
The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer.
利用表面等离子体共振技术研究了针对沙门氏菌B血清群O-多糖的特异性抗体Se155-4与配体结合的动力学。由于Fab和单链抗体可变区（scFv）制剂中痕量的寡聚物导致双相结合曲线难以分析，所有动力学测量均在纯化的单体片段上进行，对于某些突变型scFv，则在二聚体形式上进行。单体形式的结果表明，该抗体相对较低的亲和力是由于快速解离（解离速率常数koff约为0.25 s-1）。二聚体形式通常显示出约慢20倍的解离速率，结合速率常数增加5倍，达到约2×105 M-1 s-1。虽然scFv二聚体的结合阶段对单一组分相互作用模型显示出良好的曲线拟合，但解离阶段是双相的，推测是因为抗原上位点的可用性和可及性总是导致一些单价结合。二聚体的快速解离速率与单体的解离速率相同。Se155-4 IgG的解离速率与scFv二聚体观察到的非常相似，而结合速率与Fab和scFv单体获得的相同。
