Modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody.
Modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody.
复制标题
修改基于 ELISA 的亲和力测定程序：与二价抗体一起使用时需要进行校正。
DOI：
10.1016/0161-5890(87)90073-3
复制
发表时间：
1987
影响因子：
3.6
通讯作者：
Stevens,FJ
中科院分区：
文献类型：
--
作者：
Stevens,FJ
最近描述了一种评估单克隆抗体亲和力的方法[friguet al.， J. immune]。[方法77,305-319(1985)]使用ELISA系统来确定抗原和抗体混合物中存在的游离抗体的数量。然而，一个完整的IgG可以通过两个结合位点中的任何一个与抗原结合，并且无论其一个或两个结合位点是游离的，IgG都可以与固相抗原结合。因此，该程序不能直接提供配体结合位点的关注，这是计算热力学关联常数所必需的量。二项概率分布将配体结合位点的比例与未配体、单配体和双配体IgG的关注联系起来，假设每个Fab与抗原的结合是独立的。通过模拟实验比较二价IgG和单价Fab的表观结合特性，并计算两种情况下的表观结合常数。结果发现，完整IgG上结合位点的亲和力被低估了至少2倍，并且误差与配体结合位点的比例成反比。抗体的结合位点亲和力可能被低估几个数量级。在二项分析的基础上，可以将结合IgG的表观关注转化为配体结合位点的实际关注，从而在不改变实验方案的情况下计算完整IgG的有效结合常数。
A recently described procedure for the evaluation of the affinity of monoclonal antibodies [Friguetet al., J. Immun. Meth.77, 305–319 (1985)] uses an ELISA system to determine the quantity of free antibody present in a mixture of antigen and antibody. However, an intact IgG may bind antigen by either of two binding sites, and an IgG can bind to a solid-phase antigen whether one or two of its binding sites are free. Therefore, this procedure does not directly provide the concn of liganded binding sites, the quantity necessary for calculation of the thermodynamic association constant. A binomial probability distribution relates the fraction of liganded binding sites to the concn of unliganded, singly liganded, and doubly liganded IgG assuming that the binding of each Fab to antigen is independent. Simulated experiments were used to compare the apparent binding characteristics of bivalent IgG and monovalent Fab and to calculate apparent association constants in each case. It was found that the affinity of binding sites on intact IgG was underestimated by a factor of at least 2 and that the error was inversely related to the fraction of liganded binding sites. Binding site affinity of an antibody may be underestimated by several orders of magnitude. On the basis of binomial analysis, it is possible to convert apparent concns of bound IgG to actual concns of liganded binding site resulting in the calculation of valid association constants for intact IgG without alteration of the experimental protocol.