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目的：探索亲和纯化人源单克隆抗体片段的简便实用方法。方法：应用自制的抗人免疫球蛋白片段抗体（Ａｎｔｉ－Ｆａｂ）与链球菌蛋白Ｇ亲和胶（Ｇａｍ ｍ ａ Ｂｉｎｄ）交联制备亲和层析柱，用一步法从工程菌培养上清中纯化人源抗ＨＢｓ单克隆Ｆａｂ 片段。经依沙吖啶沉淀、离子交换、分子筛纯化人混合血清提取ＩｇＧ组分，木瓜酶处理后分离、纯化出Ｆａｂ，免疫绵羊制备高效价抗血清。用Ｇａｍ ｍ ａＢｉｎｄ 一步纯化出抗血清中的ＩｇＧ组分，再将该ＩｇＧ经交联剂与Ｇａｍ ｍ ａ Ｂｉｎｄ 交联后制成亲和胶。人源抗ＨＢｓ阳性菌培养上清与亲和胶共育后装柱，ＰＢＳ洗去非特异结合蛋白，再用５ ｍ ｏｌ／Ｌ氯化锂洗脱特异Ｆａｂ。结果：聚丙烯酰胺电泳纯化产物显示，纯化后的抗体片段在电泳中形成单一区带，达到电泳纯；用酶标抗Ｆａｂ 抗体免疫印迹结果证明，电泳显示的单一区带为Ｆａｂ 蛋白区带；抗原特异Ｄｏｔｂｌｏｔ检测表明，该法制备出的Ｆａｂ 保持了抗原结合活性。结论：本实验建立的一步亲和纯化法具操作简便、纯化快速和高效的特点，是人源性单克隆抗体Ｆａｂ 片段纯化的较佳方法
Objective: To explore a convenient and practical method for affinity purification of human monoclonal antibody fragments. Methods: The affinity chromatography was performed by using anti-Fab antibody and Gam-m-a-Bind. The affinity chromatography was performed on the culture supernatant of the engineered bacteria Purified human anti-HBs monoclonal Fab fragment. After the acridine acridine sedimentation, ion exchange, molecular sieve purification of human serum was extracted IgG components, papain enzyme treatment after isolation, purification of Fab, immune sheep preparation of high titer anti-serum. The anti-serum IgG fraction was purified in one step by Gam-m-aBind, and then the IgG was crosslinked with Gam-m-Bind via a cross-linker to form an affinity gel. Human anti-HBs positive culture supernatants were incubated with affinity gel and packed in PBS. Non-specific binding proteins were washed away with PBS and specific Fab was eluted with 5 mol / L lithium chloride. Results: The polyacrylamide electrophoresis purification products showed that the purified antibody fragments formed a single zone in electrophoresis and reached electrophoretic purity. The results of Western blotting with ELISA antibody showed that the single band showed by electrophoresis was Fab protein band. Antigen-specific Dotblot assay showed that the Fab prepared by this method retained the antigen-binding activity. CONCLUSION: The one-step affinity purification method established in this experiment is characterized by its simplicity, rapid purification and high efficiency. It is a better method to purify the Fab fragment of humanized monoclonal antibody