Azimzadeh A, Van Regenmortel M H
Laboratoire d'Immunochimie, Institut de Biologie Moléculaire et Cellulaire du CNRS Strasbourg, France.
J Immunol Methods. 1991 Aug 9;141(2):199-208. doi: 10.1016/0022-1759(91)90146-7.
The binding affinity of a monoclonal antibody (Mab) to tobacco mosaic virus (TMV) was determined by measuring, in an enzyme-linked immunosorbent assay, the amount of free antibody present after ultracentrifugation of virus-antibody complexes at equilibrium. In antibody excess, univalent binding of Mabs was observed and the affinity constant was K = 3.2 +/- 0.4 10(8) l/mol; in antigen excess, bivalent antibody binding was observed and the antibody avidity was about 15 times higher. In antigen excess, it was imperative to correct experimental data for the presence of 0.55% inactive molecules in the immunopurified antibody preparation. Modelling studies suggest that in the case of antibodies of increasing affinity, it becomes increasingly important to correct for the presence of inactive antibody in the binding assay.
通过在酶联免疫吸附测定中测量病毒-抗体复合物在平衡状态下超速离心后存在的游离抗体量，来确定单克隆抗体（Mab）与烟草花叶病毒（TMV）的结合亲和力。在抗体过量时，观察到Mab的单价结合，亲和常数为K = 3.2 +/- 0.4×10⁸ l/mol；在抗原过量时，观察到二价抗体结合，且抗体亲和力约高15倍。在抗原过量时，必须针对免疫纯化抗体制剂中存在0.55%无活性分子的情况对实验数据进行校正。模型研究表明，对于亲和力不断增加的抗体，在结合测定中针对无活性抗体的存在进行校正变得越来越重要。
