应用小鼠肝坏死模型分析重组全人源抗肿瘤坏死因子-α单克隆抗体的体内中和活性

Analysis of neutralizing activity in vivo of recombinant monoclonal antibody against tumor necrosis factor-α from human origin by using mouse model of liver necrosis

目的建立小鼠肝坏死模型,分析重组全人源抗肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)单克隆抗体的体内中和活性。方法确定D-半乳糖胺(D-galactosamine,D-Gal N)的致敏时间及TNF-α对C57BL/6小鼠的致死剂量;采用D-Gal N致敏小鼠,经腹腔注射TNF-α建立小鼠肝坏死模型,并测定3批供试品(抗TNF-α单抗)和3批阳性对照(阿达木单克隆抗体注射液)的体内中和活性,采用Probit回归拟合对活性测定结果进行分析。结果确定DGal N的致敏时间为10 min;TNF-α的致死剂量为100μg/kg体重;Probit回归拟合分析显示,拟合度较好,3批供试品对C57BL/6小鼠的半数有效剂量(ED50)分别为0.505、0.434和0.609 mg/kg,3批阳性对照对C57BL/6小鼠的ED50分别为0.455、0.571和0.484 mg/kg,供试品与阳性对照对C57BL/6小鼠的体内中和活性差异无统计学意义(P>0.05)。结论联合应用D-Gal N和TNF-α可造成小鼠肝坏死,从而导致小鼠死亡,抗TNF-α单抗可阻断其效应。抗TNF-α单抗对TNF-α的抑制作用呈剂量-效应关系。

Objective To establish a mouse model of liver necrosis and analyze the neutralizing activity in vivo of recombinant monoclonal antibody（Mc Ab）against tumor necrosis factor（TNF）-α from human origin. Methods The time for sensitization with D-galactosamine（D-Gal N）and lethal dose of TNF-α to C57 BL / 6 mice were determined. Mice were sensitized with D-Gal N and injected i. p. with TNF-α to establish liver necrosis model, with which the neutralizing activities in vivo of three batches of test samples（Mc Ab against TNF-α）and three batches of positive control（Humira誖）were determined, and the results were analyzed by Probit regression fitting. Results The time for sensitization with DGal N was 10 min, while the lethal dose of TNF-α was 100 μg / kg bodyweight. Probit regression fitting showed good fitting degree. The median effective doses（ED50）of three batches of test samples to C57 BL / 6 mice were 0. 505, 0. 434 and0. 609 mg / kg, while those of three batches of positive control were 0. 455, 0. 571 and 0. 484 mg / kg, respectively. The neutralizing activities in vivo of test samples and control in C57 BL mice showed no significant difference（P 〉 0. 05）.Conclusion D-Gal N combined with TNF-α caused liver necrosis, resulting in the death of mice, which could be blocked by Mc Ab against TNF-α. The Mc Ab showed dose-dependent inhibitory effect on TNF-α.