一种灵敏的酶联免疫吸附测定法（ELISA），用于检测作为固定化蛋白A纯化的鼠免疫球蛋白微量污染物存在的葡萄球菌蛋白A（SpA）。

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of staphylococcal protein A (SpA) present as a trace contaminant of murine immunoglobulins purified on immobilized protein A.

作者信息

Godfrey M A, Kwasowski P, Clift R, Marks V

机构信息

School of Biological Sciences, University of Surrey, Guildford, UK.

出版信息

J Immunol Methods. 1992 Apr 27;149(1):21-7. doi: 10.1016/s0022-1759(12)80044-5.

DOI:10.1016/s0022-1759(12)80044-5

Abstract

A specific non-competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detecting and quantifying protein A (SpA), present as a trace contaminant of therapeutic murine monoclonal antibodies (McAb) purified on immobilized SpA preparations. The assay employs a microtitration plate system, in which affinity-selected chicken anti-SpA antibodies from the egg yolks of immunized hens provide a specific capture antibody, followed by the addition of standards or sample with a McAb concentration of 1 mg/ml, in conditions unfavourable for Fc binding, and finally an affinity-selected rabbit anti-SpA peroxidase label. The working range of this assay is between 0.5 and 10.0 ng/ml (CV less than 5%), with a lower limit of detection of 0.2 ng/ml (CV less than 10%). This assay was used to evaluate SpA leakage when purifying a serum-free murine IgG1 cell culture supernatant using SpA immobilized on agarose (Protein A-Sepharose CL-4B) or controlled pore glass (Prosep A, high capacity). These gave average antibody SpA contamination levels of 6.7 +/- 1.6 and 2.4 +/- 0.5 (mean +/- SD) parts per million respectively.

摘要

已开发出一种特定的非竞争性酶联免疫吸附测定法（ELISA），用于检测和定量蛋白A（SpA），其作为在固定化SpA制剂上纯化的治疗性鼠单克隆抗体（McAb）的痕量污染物存在。该测定法采用微量滴定板系统，其中来自免疫母鸡蛋黄的亲和选择鸡抗SpA抗体提供特异性捕获抗体，随后在不利于Fc结合的条件下加入浓度为1mg/ml的McAb标准品或样品，最后加入亲和选择的兔抗SpA过氧化物酶标记物。该测定法的工作范围在0.5至10.0ng/ml之间（变异系数小于5%），检测下限为0.2ng/ml（变异系数小于10%）。该测定法用于评估使用固定在琼脂糖（蛋白A-琼脂糖CL-4B）或可控孔径玻璃（Prosep A，高容量）上的SpA纯化无血清鼠IgG1细胞培养上清液时SpA的泄漏情况。这些分别给出了平均抗体SpA污染水平为百万分之6.7±1.6和2.4±0.5（平均值±标准差）。