结核分枝杆菌蛋白抗原刺激人γδT细胞的活性单位的计算方法  

A method to calculate the bioactive unit of Mycobacterium tuberculosis antigen in stimulating human γδT cells

作　　者：孙悝[1] 陈勇[1] 徐志庆[1] 陈立[1] 李柏青[1] 

机构地区：[1]蚌埠医学院免疫学教研室安徽省感染与免疫重点实验室,233030

出　　处：《免疫学杂志》2013年第4期341-345,共5页Immunological Journal

基　　金：卫生部重大传染病专项(2012ZX10003006-004)

摘　　要：目的以检测和计算结核分枝杆菌(Mycobacterium tuberculosis,Mtb)蛋白抗原激活人γδT细胞增殖的活性单位为指标,比较不同提取分离方法制备的Mtb抗原制剂激活人γδT细胞的活性效价。方法培养的Mtb-H37Ra菌株通过高温和超声方法处理,分别获取Mtb耐热抗原(Mtb-HAg)和超声处理抗原(Mtb-SoAg),采用超滤离心管(3 K,10 K,30 K)和快速液相蛋白层析(FPLC)分组和分离,获取不同相对分子质量组分的抗原。采集健康成年人外周血分离获取PBMC,加入不同质量浓度梯度的Mtb蛋白抗原或其分离组分,并加IL-2培养12 d后荧光抗体染色,流式细胞仪检测和分析扩增T细胞中γδT细胞的比例,以扩增γδT细胞最高比例的50%(EC50)所对应的质量浓度计算为1个活性单位。结果用活性单位计算法分析发现,某批次的Mtb-HAg(591μg/ml)刺激γδT细胞扩增的EC50为1.4μg/ml,计算其活性单位为0.71 U/μg;某批次低分子Mtb-SoAg(3 K～10 K)(221μg/ml)的EC50为0.8μg/ml,其活性单位为1.25 U/μg。结论通过比较Mtb蛋白抗原不同批次及其不同组分在不同质量浓度下刺激γδT细胞扩增数量的活性,可以计算获得相应的活性单位,为检测Mtb蛋白抗原制剂刺激γδT细胞增殖活性建立了标准化的计算方法。We aimed to establish a method for detection and calculation of the bioactive unit of Mycobacterium tuberculosis （Mtb） protein antigen in stimulating human γБT cells. From cultured Mtb-H37Ra, we prepared Mtb heat resistant antigen （Mtb-HAg） and Mtb ultrasonic antigen （Mtb-SoAg） by high temperature and ultrasonic treatment respectively. By using ultra filtration centrifuge tube （3 K, 10 K, 30 K） and fast protein liquid chromatography （FPLC）, these Mtb antigens were further separated and divided into different molecular weight components. Then, PBMC from healthy adults were cultured for 12 days with different concentrations of separated components of Mtb antigens and IL-2. By using fluorescent antibody staining and flow cytometry analysis, the proportion of γБT cells among the expanded T cells was measured. The concentration of Mtb protein antigen that expanding the γБT cells to reach the 50% of the maximum proportion （ECS0） was calculated as one bioactive unit. According to the bioactive unit calculation, the ECS0 of a batch of Mtb-HAg （591 υg/ml） was 1.4 υg/ml, which was calculated as 0.71 U/υg; and the ECS0 of a batch of Mtb-SoAg with low molecular component （3 K-10 K） （221 lug/ ml） was 0.8 txg/ml, which was calculated as 1.25 U/υg of bioactive unit. By comparing the different capacity of stimulating γБT cells from different batches of the Mtb-Ag with different components and different concentrations, we could calculate the corresponding bioactive unit, which actually is a standard method for measuring the capacity of Mtb protein antigen to stimulate human γБT cells.

关 键 词：结核分枝杆菌 ΓΔT细胞 耐热抗原 超声抗原 活性单位 

分 类 号：R392.12[医药卫生—免疫学]