DIRECT MEASUREMENT OF ANTIBODY-AFFINITY DISTRIBUTION BY HAPTEN-INHIBITION ENZYME-IMMUNOASSAY
DIRECT MEASUREMENT OF ANTIBODY-AFFINITY DISTRIBUTION BY HAPTEN-INHIBITION ENZYME-IMMUNOASSAY
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DOI：
10.1016/0161-5890(84)90070-1
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发表时间：
1984-01-01
影响因子：
3.6
通讯作者：
VIVES, J
中科院分区：
文献类型：
--
作者：
NIETO, A;GAYA, A;VIVES, J
描述了一种快速、简单和可靠的技术，用于确定复杂混合物中抗体亚群的亲和力[在小鼠中]。该方法的原理是抗原浓度可以表示为固定在含有固定体积稀释抗体的微孔聚苯乙烯表面的抗原量。通过测量抗体结合到涂有不同数量抗原的不同孔的比例，计算亲和分布是一件简单的事情。结合在聚苯乙烯板上的抗原量与用于致敏的抗原浓度成正比，并遵循典型的饱和曲线。结合在低浓度抗原致敏板上的抗体比结合在高浓度抗原致敏板上的抗体具有更高的亲和力。表观亲和常数（aK）定义为50%抑制抗体与固定抗原结合所需的游离半抗原的倒数浓度。该方法测定的aK值与荧光猝灭法或法尔法测定的本征亲和常数K值吻合较好。在体内免疫过程中，有一个明显的向高亲和力抗体亚群的转变。
A rapid, simple and reliable technique for determining the affinities of antibody subpopulations in a complex mixture is described [in the mouse]. The principle of this method is that antigen concentration can be represented as the amount of antigen immobilized on the polystyrene surface of a microwell containing a fixed volume of diluted antibody. By measuring the proportion of antibody bound to different wells coated with varying amounts of antigne, it is a straightforward matter to calculate an affinity distribution. The amount of antigen bound to a polystyrene plate is proportional to the concentration of antigen used for sensitization and follows a typical saturation curve. The antibodies bound to plates sensitized with low concentrations of antigen are of higher affinity than those bound to plates sensitized with high concentrations of antigen. An apparent affinity constant (aK) is defined as the reciprocal concentration of free hapten required for 50% inhibition of antibody binding to immobilized antigen. The aK determined by this method is in close agreement with the intrinsic affinity constant (K) measured by fluorescence quenching or the Farr assay. During the course of immunization in vivo there is a clear shift to higher-affinity antibody subpopulations.