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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。
OBJECTIVE To detect and calculate the activity units of Mycobacterium tuberculosis (MTb) protein antigen-activated human γδT cell proliferation as an index to compare the activity titers of Mtb antigen preparations activated by different extraction and isolation methods. Methods The Mtb-H37Ra strain was cultured and treated with hyperthermia and sonication. Mtb-HAg and Mtb-SoAg were obtained by ultracentrifugation (3 K, 10 K, 30 K) And fast liquid phase protein chromatography (FPLC) were grouped and isolated to obtain antigens of different relative molecular weight components. Peripheral blood of healthy adults were collected to obtain PBMC, and different concentrations of Mtb protein antigen or its fractions were added into the peripheral blood of healthy adults. Fluorescent antibody staining was also performed after 12 days of culture with IL-2. Flow cytometry was used to detect and analyze the expanded T cells The ratio of γδT cells is calculated as 1 unit of activity corresponding to the mass concentration corresponding to the 50% (EC50) of the highest proportion of γδT cells amplified. Results The results showed that the EC50 of a batch of Mtb-HAg (591μg / ml) stimulated γδT cell expansion was 1.4μg / ml and the activity unit was calculated to be 0.71 U / μg. A batch of low-molecular-weight Mtb The EC50 of -SoAg (3 K ~ 10 K) (221 μg / ml) is 0.8 μg / ml with an activity unit of 1.25 U / μg. Conclusion By comparing the activity of different batches of Mtb antigen and its different components in stimulating the number of γδT cells under different mass concentrations, the corresponding active units can be calculated and established to test the Mtb protein antigen preparation to stimulate the proliferation activity of γδT cells The calculation method.