低温乙醇法和硫酸铵盐析法制备的抗人T细胞猪免疫球蛋白与同类型兔源性制品体外活性的比对  

In vitro activity comparison between anti-human T lymphocyte porcine immunoglobulin prepared by Cohn ethanol fractionation and ammonium sulphate precipitation and same type of rabbit-derived products

作　　者：杨帆 杜洪桥 邹浩勇 王智 薛红刚 黄梦 闫新业 张智 YANG Fan;DU Hongqiao;ZOU Haoyong;WANG Zhi;XUE Honggang;HUANG Meng;YAN Xinye;ZHANG Zhi(不详;Process Research Department,Yujin Bio-Pharma Wuhan CNBG Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉中生毓晋生物医药有限责任公司工艺研究项目组,湖北武汉430207 [2]武汉生物制品研究所有限责任公司科研开发部,湖北武汉430207

出　　处：《中国生物制品学杂志》2024年第6期687-695,702,共10页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2020YFC0841800)。

摘　　要：目的比较低温乙醇法和硫酸铵盐析法制备的抗人T细胞猪免疫球蛋白(anti-human T lymphocyte porcine immunoglobulin,P-ATG)与市售的抗人胸腺细胞及抗人T细胞兔免疫球蛋白的体外活性,以评价两种工艺制备的P-ATG的性能。方法采用乳酸脱氢酶(lactate dehydrogenase,LDH)法检测4种制品杀伤靶细胞的抗体依赖细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)效应;CCK-8法检测4种制品杀伤靶细胞的补体依赖性细胞毒作用(complement dependent cytotoxicity,CDC)效应;细胞双重免疫荧光标记法检测4种制品与不同T细胞抗原(CD3、CD4、CD8)的共定位情况。结果4种制品中,仅抗人胸腺细胞兔免疫球蛋白在高浓度(1 mg/mL)下对人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)有较弱的ADCC效应;4种制品均可介导人PBMC产生CDC效应,且呈剂量依赖性,其中抗人胸腺细胞兔免疫球蛋白的活性较高,为两种工艺制备的P-ATG或抗人T细胞兔免疫球蛋白的3~4倍,抗人T细胞兔免疫球蛋白的活性与P-ATG相当;4种制品与T细胞表面抗原CD3、CD4的共定位细胞比例相近,抗人胸腺细胞及抗人T细胞兔免疫球蛋白与CD8抗原共定位的细胞比例略低于两种工艺制备的P-ATG。结论低温乙醇法与硫酸铵盐析法制备的P-ATG体外活性具有良好的一致性,且在临床剂量下不低于进口同类型制品。Objective To compare the in vitro activity of anti-human T lymphocyte porcine immunoglobulin(P-ATG)prepared by Cohn ethanol fractionation and ammonium sulphate precipitation,commercial rabbit anti-human thymocyte immunoglobulin(trade name:Thymoglobuline),and anti-human T lymphocyte rabbit immunoglobulin(trade name:Grafalon)so as to evaluate the properties of P-ATG prepared by two processes.Methods The four products were detected for the antibody-dependent cell-mediated cytotoxicity(ADCC)by lactate dehydrogenase(LDH)method,measured for the complement dependent cytotoxicity(CDC)by CCK-8 assay,and detected for the affinity for binding with different T cell antigens(CD3,CD4,CD8)by double immunofluorescence staining method.Results Among the four products,only Thymoglobuline at the high concentration(1 mg/mL)had a weak ADCC effect on human peripheral blood mononuclear cell(PBMC).All products could induce the CDC effect in human PBMC in a dose-dependent manner,among which the effect of Thymoglobuline was higher than that of P-ATG or Grafalon preared by two processes,about 3 to 4 times,and the effect of Grafalon was comparable to that of P-ATG.In the proportion of four products binding with T cell surface antigens CD3 and CD4 was similar,However the proportion of Thymoglobuline and Grafalon binding with CD8 antigen was slightly lower than that of P-ATG.Conclusion The in vitro activity of the P-ATG prepared by Cohn ethanol fractionation and ammonium sulphate precipitation was in good agreement,and was not lower than that of imported products at the clinical dose.

关 键 词：低温乙醇法 硫酸铵盐析法 抗人T细胞免疫球蛋白 体外活性 

分 类 号：R392-33[医药卫生—免疫学]