Cho E W, Lee M K, Kim K L, Hahm K S
Protein Engineering Research Group, Genetic Engineering Research Institute, KIST, Taejon, Korea.
J Immunoassay. 1995 Nov;16(4):349-63. doi: 10.1080/15321819508013567.
A simple method for determination of binding kinetics of a solid-phase antibody using antigen-beta-galactosidase hybrid protein was evaluated. To minimize conformational change of the antigen binding site of the antibody when directly binding to a microtiter plate, the microtiter plate was precoated with protein A. The binding and free antigen concentrations were directly obtained from the beta-galactosidase activity. This method can be used for analyses of the equilibrium dissociation constant (KD), and the association (Kass) and dissociation (Kdiss) rate constants. Peptide antigenicity was also analyzed by competitive ELISA using this method. Since both antigen-beta-galactosidase and the peptide used are localized in the fluid-phase, the proper affinity constant (KA) of the peptide can be estimated from the KD value of the antigen-beta-galactosidase-antibody interaction, and from the IC50 value of the peptide.
评估了一种使用抗原-β-半乳糖苷酶杂交蛋白测定固相抗体结合动力学的简单方法。为了在抗体抗原结合位点直接与微量滴定板结合时尽量减少其构象变化，微量滴定板预先用蛋白A包被。结合抗原和游离抗原的浓度可直接从β-半乳糖苷酶活性获得。该方法可用于分析平衡解离常数（KD）、缔合（Kass）和解离（Kdiss）速率常数。还使用该方法通过竞争性ELISA分析了肽的抗原性。由于抗原-β-半乳糖苷酶和所用肽均存在于液相中，因此可以从抗原-β-半乳糖苷酶-抗体相互作用的KD值以及肽的IC50值估算肽的适当亲和常数（KA）。
