Pauly J U, Siebold B, Schulz R, List W, Lüben G, Seiler F R
Research Laboratories, Behringwerke AG, Marburg, W.-Germany.
Behring Inst Mitt. 1990 Oct(86):192-207.
An enzyme linked immunosorbent assay (ELISA) has been developed for the quantitative determination of the most probable contaminating proteins (MCP) of recombinant human Erythropoietin produced in the mouse fibroblast cell line C-127. The developed ELISA is a double polyclonal sandwich type immunoassay, which allows a quantitation of the MCPs in the range of parts per million. The polyclonal antibody, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference MCPs (a collection of the most probable protein contaminants) by immunoblot analysis and immunoabsorption of radiolabeled MCPs. Affinity purification of this antibody preparation using the immobilized MCPs resulted in an assay with higher signal-to-noise ratio. The assay was demonstrated to be very specific for the MCPs obtained from the rhu EPO purification process. Since the purification of each recombinant DNA derived protein expressed in mammalian cells requires its own unique process, no generic assay for contaminating proteins can be developed. There are only a few common criteria for the development of such multi-antigen ELISA, which will be discussed in this paper.
已开发出一种酶联免疫吸附测定（ELISA）法，用于定量测定在小鼠成纤维细胞系C-127中生产的重组人促红细胞生成素最可能的污染蛋白（MCP）。所开发的ELISA是一种双多克隆夹心型免疫测定法，可对百万分之几范围内的MCP进行定量。在该ELISA中用于包被和缀合的多克隆抗体，通过免疫印迹分析和放射性标记MCP的免疫吸附，被证明与参考MCP（最可能的蛋白质污染物集合）具有反应性。使用固定化MCP对该抗体制剂进行亲和纯化，得到了信噪比更高的测定法。该测定法被证明对从重组人促红细胞生成素纯化过程中获得的MCP具有高度特异性。由于在哺乳动物细胞中表达的每种重组DNA衍生蛋白的纯化都需要其独特的过程，因此无法开发通用的污染蛋白测定法。本文将讨论开发这种多抗原ELISA的几个共同标准。
