用于小鼠IgE的特拉萨基酶联免疫吸附测定法。III. 通过序列平衡结合分析测定浓度和功能亲和力。

Terasaki-ELISA for murine IgE. III. Determination of concentration and functional affinity by sequential equilibrium binding analysis.

作者信息

Pathak S S, Vos Q, Savelkoul H F

机构信息

Department of Immunology, Erasmus University, Rotterdam, The Netherlands.

出版信息

J Immunol Methods. 1989 Sep 29;123(1):71-81. doi: 10.1016/0022-1759(89)90031-8.

DOI:10.1016/0022-1759(89)90031-8

PMID:2794530

Abstract

A simple Terasaki tray-based ELISA technique with a fluorescent detecting system has been used to determine the affinity of murine IgE antibodies. The system was shown to be sensitive enough to measure affinities in the range of 10(-6)-10(-10) M as well as detect IgE antibodies down to a limit of 0.1 ng/ml. The results, expressed as arbitrary fluorescence units (AFU), were compared with those obtained using equilibrium dialysis for several DNP-specific IgE monoclonal antibodies of known affinities yielding KD values. The relationship between KAFU and KD established a conversion factor which could then be used to compute KD from KAFU, provided the detection system remained identical. Based on the equations proposed, an alternative method for the quantitation of murine IgE is described which is independent of the affinity of IgE for the coated antigen.

摘要

一种基于Terasaki板的带有荧光检测系统的简单酶联免疫吸附测定（ELISA）技术已被用于测定小鼠IgE抗体的亲和力。该系统被证明灵敏度足以测量10^(-6)-10^(-10) M范围内的亲和力，并且能够检测低至0.1 ng/ml的IgE抗体。结果以任意荧光单位（AFU）表示，并与使用平衡透析法对几种已知亲和力（产生KD值）的DNP特异性IgE单克隆抗体所获得的结果进行比较。KAFU与KD之间的关系建立了一个转换因子，只要检测系统保持相同，该转换因子随后可用于从KAFU计算KD。基于所提出的方程，描述了一种定量小鼠IgE的替代方法，该方法与IgE对包被抗原的亲和力无关。