【摘要】 提出一种简便的免疫亲和层析方法，有效地分离纯化重组人肿瘤坏死因子（ｒＨＴＮＦ），此法不需预先分离纯化单克隆抗体．用交联刘ｄｉｍｅｔｈｙｌｓｕｂｅｒｉｍｉｄａｔｅ使抗ＴＮＦ的单克隆重抗体和ｐｒｏｔｅｉｎＡ—ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ共价结合，成为稳定的免疫基质，制备免疫亲和层析柱，将重组ＴＮＦ粗制品一步纯化为均一组份．更多还原

【Abstract】 A single-step purification procedure yielding homogeneity is desribed. The new method provides a highly specific immunomatrix with stable linking without purificahon of monoclonal antibody. A homologous preparation from crude extracts of TNF-a can be purified in a single step with nearly quantitative yield.Previously conventional immunoaffinity chromatography has proved a potent method of protein purification. However, the monoclollal antibody was needed to purify to homogeneity, then the purified antibody was coupled to CNBr-activated Sepharose. The method presented here demonstrates that the antibody has been coupled to protein A Sepharose CL-4B instead of CNBr-activated Sepharose. So toxic agent CNBr is not involved. In new coupling systenl, the Fo fractiolls of monoclonal antiboby are covalently coupled to protein A Sepharose Cl-4B using dimethylsuberimidate as a cross-linking agent. In this case, the Fab fractions of antibody molecules can be maintained free. and bound with TNF-a. This provides some advantage for more antigens binding resulting higher yield.Our results show that use of this system is simpler, faster and higher yield compared with the comomly used CNBr-activated Sepharose. For example, l.5mg of purified TNF-a can be obtained from ammonia sulfate precipitated fractions on a 5ml immunoaffinity chromatography column which is coupled with 10mg of monoclonal antibody from 3ml of ascitic fluids, wereas about ling of puified TNF-a is isolated by CNBr-activated coupled gel using the same amount of monoclonal antibody. The purity and biological activity of the purified TNF-a is determined by SDS-PAGE and by mouse L-929 cells killing activity. This TNF-a shows a single protein band at an apparent molecular weight of 17,000 by SDS-PAGE under reducing conditions; the specific activity is 2×107units/mg. The above results indicate that the purified TNF-a is identical with the Stantard TNF-a.更多还原