Rabbany S Y, Piervincenzi R, Judd L, Kusterbeck A W, Bredehorst R, Hakansson K, Ligler F S
Bioengineering Program, Hofstra University, Hempstead, New York 11550, USA.
Anal Chem. 1997 Jan 15;69(2):175-82. doi: 10.1021/ac960680h.
The intrinsic binding characteristics of monoclonal antibodies are modified upon immobilization onto a solid-phase matrix. Factors such as the distribution in affinity must therefore be taken into consideration in order to predict the kinetics of antibody binding at solid-liquid interfaces. A mathematical analysis is presented herein that allows the assessment of heterogeneity in the affinity of monoclonal antibodies immobilized onto a solid support. This model is based on a modified version of the Sips distribution function adapted to the conditions of a solid-phase displacement assay in flow. An assay for trinitrotoluene (TNT) provides the data to evaluate the extent of heterogeneity introduced by immobilization of antibodies in a flow immunoassay. We determined the index of antibody heterogeneity on two solid supports, controlled-pore glass beads and agarose beads, coated with a monoclonal anti-TNT antibody at varying densities. The data confirm that the threshold for crossover from homogeneous to heterogeneous forms of the reaction isotherm is different in displacement reactions than in association-dissociation reactions. Our analysis shows that the measured displacement isotherm is consistent with a homogeneous or only moderately heterogenous distribution of relative affinities.
单克隆抗体固定在固相基质上后，其内在结合特性会发生改变。因此，为了预测抗体在固液界面的结合动力学，必须考虑诸如亲和力分布等因素。本文提出了一种数学分析方法，可用于评估固定在固相支持物上的单克隆抗体亲和力的异质性。该模型基于对Sips分布函数的改进版本，适用于流动相中固相置换分析的条件。三硝基甲苯（TNT）分析提供了数据，以评估在流动免疫分析中抗体固定化引入的异质性程度。我们测定了在不同密度下涂覆有单克隆抗TNT抗体的两种固相支持物（可控孔径玻璃珠和琼脂糖珠）上抗体的异质性指数。数据证实，在置换反应中，反应等温线从均相转变为异相形式的阈值与缔合-解离反应中的不同。我们的分析表明，测得的置换等温线与相对亲和力的均相或仅中等程度的异质分布一致。
