纯化植物病毒间接酶联免疫吸附测定中底物水解速率的分析与应用

Analysis and application of substrate hydrolysis rates in indirect ELISA of a purified plant virus.

作者信息

Vilker V L, Verduin B J

机构信息

Department of Chemical Engineering, University of California, Los Angeles 90024.

出版信息

J Virol Methods. 1988 Feb;19(2):141-9. doi: 10.1016/0166-0934(88)90157-7.

DOI:10.1016/0166-0934(88)90157-7

PMID:3366853

Abstract

The transient colorimetric signal in a microtiter plate is used to quantify a purified plant virus, cowpea mosaic virus (CPMV), over five concentration decades in a single plate. The method involves the coating of the polystyrene microtiter plate wells directly with the CPMV antigen, followed by incubation with a rabbit-derived CPMV-specific antibody, and lastly by incubation with a commercially available antibody against rabbit immunoglobulin which has been pre-labeled with alkaline phosphatase. The rate of p-nitrophenylphosphate hydrolysis, both non-specific and that which was catalyzed by this enzyme, was measured spectrophotometrically at 405 nm. Enzyme-catalyzed hydrolysis rates followed first order kinetics at all antigen coating concentrations, and the 1 degree rate constants, which ranged from 2 X 10(-6) min-1 to 1 X 10(-3) min-1, were found to increase with increasing antigen concentration.

摘要

微量滴定板中的瞬态比色信号用于在单个板中对纯化的植物病毒豇豆花叶病毒（CPMV）在五个浓度数量级上进行定量。该方法包括直接用CPMV抗原包被聚苯乙烯微量滴定板孔，然后与兔源CPMV特异性抗体孵育，最后与已用碱性磷酸酶预标记的抗兔免疫球蛋白的市售抗体孵育。在405nm处通过分光光度法测量对硝基苯磷酸酯的非特异性水解速率以及该酶催化的水解速率。在所有抗原包被浓度下，酶催化的水解速率均遵循一级动力学，并且发现1级速率常数范围为2×10^(-6) min^(-1)至1×10^(-3) min^(-1)，随抗原浓度的增加而增加。