免疫亲和层析纯化EGF-PE40

Purification of EGF-PE40 by Immunoaffinity Chromatography

用 PEG融合法建立了分泌抗 EGF- PE4 0抗体的杂交瘤细胞株 ,并筛选了一杂交瘤细胞株 D7。用此细胞株制备了高抗体含量的腹水 ,用辛酸 -硫酸胺沉淀法纯化了抗体 Ig G,用 EL ISA法测定其与 EGF- PE4 0的亲和常数 2 .2 4×1 0 9L / mol,并鉴定其亚类为 Ig G1。将纯化后的单抗 Ig G与 CNBr活化的 Sepharose 4 B偶联 ,并用免疫亲和层析的方法纯化粗提物中的 EGF- PE4 0 ,得到了大于 80 %的纯化率 ,且产物显示了良好的生物学活性 ,这为 EGF- PE4

The hybridoma cell line secreting antibody against EGF PE40 was constructed by techniques of cell fusion with polyethylene glycol (PEG) and screened a secreting cell line, D 7. Ascits of high concentration of McAb were prepared. After one McAb being purified from ascits, the affinity constant of the McAb and EGF PE40 was determined by ELISA (2.24×10 9 L/mol), and its class and subclass were determined also (IgG1). Purified McAb was coupled to CNBr activated Sepharose 4B and subsequently used to purify EGF PE40 from the crude extract. The purity of EGF PE40 purified by this immunoaffinity chromatography method was over 80% higher than that of crude extract. And the purified products activated to cancer cells overexpressing EGFR. This report was significant for the further purification and production of EGF PE40.