【摘要】 目的　探讨影响酶联免疫斑点 (ELISPOT)实验特异性及敏感性的因素 ,优化酶联免疫斑点法检测抗原特异的CTL免疫应答的实验条件。方法　HLA A2阳性正常人的外周血单个核细胞在体外经流感病毒抗原肽诱导 1周后 ,观察不同实验条件对酶联免疫斑点法检测抗原特异的CTL免疫应答的影响。结果　CTL诱导时 ,效应细胞先分离出CD8+ T细胞组获得的Flu抗原特异的细胞频数要高于CTL诱导后再分离出CD8+ T细胞组 (P <0 .0 5 ) ;自体DC(树突状细胞 )作APC(抗原提呈细胞 )比自体CD8- PBMC作APC ,CTL的诱导效率高且特异 (P <0 .0 5 ) ;使用细胞因子组合 (IL 7+IL 2 +IL 6 )优于单纯使用IL 2。CTL行ELISPOT检测时 ,T2 2细胞较T2 1细胞具有更强抗原提呈功能 ,增加Flu抗原特异的CD8+ T细胞频数 (P <0 .0 5 )。结论　优化了酶联免疫斑点法检测抗原特异的CTL免疫应答的试验条件 ,优化后的方法在临床肿瘤疫苗的研究中具有应用价值更多还原

【Abstract】 Objective To define the factors which affect the specificity and sensitivity for detecting the antigen specific CTL immune response in ELISPOT assay, so as to optimize the ELISPOT assay. Methods PBMC derived CD8 + T cells specific to HLA A*0201 binding peptide influenza matrix peptide Flu p58 66 were induced in vitro 7 days′ stimulation with peptide pulsed irradiated PBMCs or DCs from HLA A2 positive healthy donors, the frequency of effector cells to secrete IFN gamma in response to Flu p58 66 was detected with an improved ELISPOT assay. Results The frequency of effector cells to secrete IFN gamma in response to Flu peptide can be increased when the CD8 + T cells were separated from PBMC before antigen stimulation than the CD8 + T cells were separated from PBMC after antigen stimulation ( P <0.05). For CTL activation, DCs was better than CD8 - PBMCs as APC ( P <0.05); the cytokine cocktail (IL 7+IL 2+IL 6) was more effective than the IL 2 alone for CTL proliferation. During the CTL functional monitoring in ELISPOT assay, T2 2 cells were more potent than that of T2 1 cells to stimulate effector cells, the former reflect to stimulate effector cells to generate more numbers of specific spots ( P <0.05). Conclusion When the critical factors are optimized, the specificity and sensitivity of IFN gamma producing ELISPOT assay can be substantially improved.更多还原