Anicetti V R, Fehskens E F, Reed B R, Chen A B, Moore P, Geier M D, Jones A J
J Immunol Methods. 1986 Jul 24;91(2):213-24. doi: 10.1016/0022-1759(86)90481-3.
An enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of part-per-million levels of the most probable E. coli polypeptide (ECP) contaminants of E. coli produced biosynthetic human growth hormone (hGH). The antibody preparation, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference ECPs (a collection of the most probable protein contaminants) by both affinity chromatography and immunoblot analysis. Affinity purification of this antibody preparation using immobilized reference ECPs resulted in an assay with a higher signal-to-noise ratio and also 'normalized' the antibody population to approach stoichiometric equivalence with the immobilized ECPs. Reference ECPs, size fractionated by gel filtration, were quantitated in agreement with their absorbance at 280 nm. The assay was demonstrated to be specific for ECPs obtained from the hGH purification process. Since the purification of each recombinant DNA derived protein from E. coli requires its own unique process, this means that no generic ECP assay can be developed. It is felt that the criteria established for this assay provide a comprehensive approach to the development of quantitative multiple antigen immunoassays.
已开发出一种酶联免疫吸附测定法（ELISA），用于定量检测大肠杆菌产生的生物合成人生长激素（hGH）中百万分之一水平的最可能的大肠杆菌多肽（ECP）污染物。在该ELISA中用于包被和缀合的抗体制剂，通过亲和色谱法和免疫印迹分析证明与参考ECP（最可能的蛋白质污染物集合）具有反应性。使用固定化参考ECP对该抗体制剂进行亲和纯化，得到了具有更高信噪比的测定法，并且还“标准化”了抗体群体，使其与固定化ECP接近化学计量当量。通过凝胶过滤进行大小分级的参考ECP，其定量结果与它们在280nm处的吸光度一致。该测定法被证明对从hGH纯化过程中获得的ECP具有特异性。由于从大肠杆菌中纯化每种重组DNA衍生的蛋白质都需要其自身独特的过程，这意味着无法开发通用的ECP测定法。人们认为，为该测定法建立的标准为定量多抗原免疫测定法的开发提供了一种全面的方法。
