MEASUREMENTS OF THE TRUE AFFINITY CONSTANT IN SOLUTION OF ANTIGEN-ANTIBODY COMPLEXES BY ENZYME-LINKED IMMUNOSORBENT-ASSAY
MEASUREMENTS OF THE TRUE AFFINITY CONSTANT IN SOLUTION OF ANTIGEN-ANTIBODY COMPLEXES BY ENZYME-LINKED IMMUNOSORBENT-ASSAY
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DOI：
10.1016/0022-1759(85)90044-4
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发表时间：
1985-01-01
影响因子：
2.2
通讯作者：
GOLDBERG, ME
中科院分区：
文献类型：
--
作者：
FRIGUET, B;CHAFFOTTE, AF;GOLDBERG, ME
本文介绍了一种测定溶液中抗原-抗体平衡解离常数(Kd)的简单、通用的方法。首先，将单抗与抗原在溶液中孵育，直到达到平衡；然后用经典的间接ELISA[酶联免疫吸附试验]来测量在平衡时保持不饱和的抗体的比例。用该方法测得的2株单抗的Kd值与常规方法(标记抗原的免疫沉淀法或荧光转移法)非常接近。只要在总抗原浓度远远超过总抗体浓度的条件下进行测量，即使用粗制的单抗也能测定抗体-抗原复合体的解离常数。该方法灵敏度高，可检测极小浓度的抗体，并可测定小至10-9M的Kd值。由于不需要标记抗原或抗体，该方法还具有处理未修饰分子的巨大优势。
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 [enzyme-linked immunosorbent assay]. The experimental values of Kd found by this ELISA procedure for 2 monoclonal antibodies are very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen or fluorescence transfer). 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 the antigen or the antibody is required.