建立结核分枝杆菌抗原K6IFN-γ ELISPOT用于结核病辅助诊断  

Establishing Mycobacterium tuberculosis antigen K6 IFN-γ ELISPOT for diagnosis tuberculosis

作　　者：蒋丽气[1] 黄香玉 陈红兵[3] 李静[3] 何珂[3] 邵进士[1] 张春青[1] 何秀云 

机构地区：[1]西南大学生命科学学院,重庆400715 [2]解放军第三0九医院器官移植与免疫调节北京市重点实验室,北京100091 [3]解放军第三0九医院结核病研究所,北京100091

出　　处：《中国免疫学杂志》2013年第6期600-604,609,共6页Chinese Journal of Immunology

基　　金：国家重大传染病专项(2013ZX10003006-003-001)

摘　　要：目的:建立结核分枝杆菌抗原K6作为刺激源的IFN-γELISPOT检测方法,并初步评价该方法用于结核病辅助诊断价值。方法:结核病患者和非结核性肺部相关疾病患者外周血单核细胞(PBMC)分别加入预包被ELISPOT板对应孔,分别加入抗原K6、T-SPOT.TB试剂盒抗原A和B,每个PBMC样品设阴性对照孔和阳性质控孔。37℃、5%CO2培养20～24小时,弃细胞,依次加入酶标检测抗体、底物显色获得斑点。ELISPOT读板仪进行斑点计数和分析。结果:K6和T-SPOT.TB试剂盒检测敏感度分别为80.0%和85.9%、特异度分别为83.9%和73.2%,两者比较差异均无统计学意义。K6作为刺激原的IFN-γELISPOT诊断结核病阳性预示值为88.3%,阴性预示值为73.4%。结核病组,抗原B刺激PBMC分泌IFN-γ的细胞斑点数(SFC)平均值分别高于抗原A和K6(P=0.011和P=0.004),抗原A和K6刺激PBMC分泌IFN-γ的SFC平均值比较差异无统计学意义(P>0.05)。非结核性肺部相关疾病组,抗原A、抗原B和K6刺激PBMC分泌IFN-γ的SFC平均值两两比较差异均无统计学意义(P>0.05)。结论:K6可作为结核病IFN-#ELISPOT诊断候选抗原之一。Objective ：To establish IFN-γ ELISPOT for antigen K6 of Mycobacterium tuberculosis in order to evaluate it as diagnosis tuberculosis. Methods： The peripheral blood mononuclear cells （PBMC） from tuberculosis （TB） and nontuberculous pulmonary disease patients were added to precoated plates. In-house antigen K6, T-SPOT. TB antigen A and antigen B were added to correspond- ing wells including negative and positive control for each sample, respectively. The plates were incubated overnight in a 37~C humidi- fied incubator with 5% CO2. The cells were discarded and the plates were washed with PBS for six times. The detection antibody con- jugated with alkaline phosphatase was diluted and added 50 μl/well at 2-8℃ for one hour. The plates were washed with PBS for six times. 50μl BCIP-NBT substrate was added to each cell. The plates incubated at room temperature for seven minutes and washed with ddH20. Spot forming cells （SCF） were detected with CTL ImmunoSpot analyzers. Results： The sensitivity and specificity of antigen K6 and T-SPOT. TB were 80.0% and 83.9% , 85.9% and 73.2% , respectively. There were no significant difference in sensitivity and specificity between antigen K6 and T-SPOT. TB. The positive and negative predictive value of IFN-γ ELISPOT for antigen K6 was 88.3% and 73.4% , respectively. For TB patients, the median SCF numbers was higher for antigen B as stimulation than antigen A and K6 as stimulation （ P = 0.011 and P =0. 004）, respectively. There was no significant difference in median SCF secreting IFN-γ in PBMC stimulated by antigen A and K6 （P 〉 0.05）. For nontuberculous pulmonary disease, there was no significant difference in median number of SCF secreting IFN-γ in PBMC stimulated by antigen A, B and K6. Conclusion： Antigen K6 may be a candidate antigen for diagnosis TB using IFN-γ ELISPOT.

关 键 词：结核病 抗原K6 ELISPOT IFN-Γ 

分 类 号：R392-33[医药卫生—免疫学]