Friguet B, Chaffotte A F, Djavadi-Ohaniance L, Goldberg M E
J Immunol Methods. 1985 Mar 18;77(2):305-19. doi: 10.1016/0022-1759(85)90044-4.
A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.
本文描述了一种简单通用的方法，用于测定溶液中抗原 - 抗体平衡的解离常数（KD）。首先，将单克隆抗体与抗原在溶液中孵育，直至达到平衡；然后，通过经典的间接酶联免疫吸附测定法（ELISA）测量在平衡时仍未饱和的抗体比例。通过这种ELISA方法测得的两种单克隆抗体的KD实验值，与通过传统方法（放射性标记抗原的免疫沉淀或荧光转移）获得的值非常接近。此外，研究表明，只要在总抗原浓度大大超过总抗体浓度的条件下进行测量，即使使用粗制的单克隆抗体制剂，也可以测定抗体 - 抗原复合物的解离常数。所使用的ELISA的灵敏度允许检测极低浓度的抗体，并测定低至10^(-9) M的KD值。该方法还具有处理未修饰分子的巨大优势，因为无需对抗原或抗体进行标记。
