通过椭偏测量法测定单克隆抗体的亲和力。

Determination by ellipsometry of the affinity of monoclonal antibodies.

作者信息

Nygren H, Kaartinen M, Stenberg M

出版信息

J Immunol Methods. 1986 Sep 27;92(2):219-25. doi: 10.1016/0022-1759(86)90169-9.

Abstract

The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies.

摘要

通过椭偏测量法研究了单克隆抗体与表面固定化半抗原之间的反应，该方法可对蛋白质的表面浓度进行绝对测量。使用了针对2-苯基恶唑酮的单克隆抗体，并通过根据测量抗体结合导致的半抗原荧光猝灭所获得的数据计算平衡常数，来确定它们在溶液中对抗原的亲和力。通过椭偏测量法测量了抗体与表面固定化半抗原的结合速率以及复合物的解离速率。通过Scatchard图确定了异质抗原-抗体反应的平衡常数。发现抗体在异质反应中对抗原的亲和力比在同质反应中高，不同单克隆抗体的该因子有所不同。