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TNT-1是-种鼠IgG_2a单抗,已用于肿瘤病人的临床试验。本文将介绍从腹水中进一步纯化TNT-1的方法及制备其F(ah′)_2片段的条件。经Protein A亲和柱层析从小鼠腹水中提取出的lgG,再通过阳离子交换树脂柱Mono S,用缓冲液A(20 mmol/L MES,pH6.3)和缓冲液B(lmol/L NaCI),在快速蛋白液相色层(FPLC)系统上进行梯度洗脱分离,可制得纯净的TNT-1抗体,其免疫活性可达80％～85％;经纯化的TNT-1,用胃蛋白酶消化,制备F(ah′)~2片段,其消化条件是:酶的用量为抗体的1/250,pH3.8,温度37℃,时间4～5 /小时。消化后的混合物,用protein A亲和结合法除去完整的IgG,其余的F(ab′)_2和Fab、Fc等片段,在Mono S柱上,按前述的条件分离。产物TNT-1或F(ah′)_2的免疫活性,用标记抗体和过量抗原结合的方法测定;分高纯化过程中的洗脱峰,用间接免疫荧光技术监测其活性。结果戾明,TNR-1的F(ab′)_2片段的制备是成功的,色层分离纯化的方法简便而有效,并已成功地应用于批量生产。
TNT-1 is a murine IgG2a monoclonal antibody that has been used in clinical trials in cancer patients. This article describes the method for the further purification of TNT-1 from ascites and the conditions for the preparation of its F (ah ’) _2 fragment. The lgG extracted from mouse ascites by Protein A affinity column chromatography was passed through a cation exchange resin column Mono S and eluted with buffer A (20 mmol / L MES, pH 6.3) and buffer B (1 mol / L NaCI ), And the purified TNT-1 antibody was purified by gradient elution on a fast protein liquid chromatography (FPLC) system with an immunogenicity of 80% ~ 85%. The purified TNT-1 Protease digestion, F (ah ’) ~ 2 fragment, the digestion conditions are: the amount of enzyme antibody 1/250, pH3.8, temperature 37 ℃, time 4 ~ 5 / hour. After digestion of the mixture, intact IgG was removed by protein A affinity and the remaining F (ab ’) 2 and Fab, Fc fragments were separated on the Mono S column by the above conditions. The immunogenicity of the product TNT-1 or F (ah ’) _2 was determined by the combination of labeled antibody and excess antigen. The elution peak during high-purification was monitored by indirect immunofluorescence assay. As a result, the preparation of F (ab ’) _ 2 fragment of TNR-1 was successful. The chromatographic separation and purification method was simple and effective, and has been successfully applied to mass production.