目的  优化酶联免疫斑点试验（enzyme-linked immunospot assay，ELISPOT）方法，提高其在重组天坛株痘苗病毒艾滋病疫苗细胞免疫评价中的灵敏度。方法  将疫苗或对照痘苗病毒皮内免疫BALB/c小鼠，50 μl/只，6周后无菌采集小鼠脾脏。用ELISPOT对分泌IFN-γ的脾淋巴细胞〔斑点形成细胞（spot-forming cell，SFC）〕进行检测。比较不同脾淋巴细胞密度（2×10⁵、1×10⁶、2×10⁶个/孔）和不同刺激肽质量浓度（2、4 μg/ml）对SFC的影响。分别采用单因素方差分析和t检验进行多组间和两组间数据比较。结果  3个细胞密度组（F=0.023，P>0.05）和2个刺激肽浓度组（t=-1.762~-0.110，P值均>0.05）间的SFC数差异均无统计学意义。当细胞密度为2×10⁵个/孔、刺激肽质量浓度为4 μg/ml时，疫苗组的SFC数达到（107.00±42.01）个/106细胞，高于原实验条件（细胞密度为1×10⁶个/孔、刺激肽质量浓度为2 μg/ml）的SFC数〔（76.55±64.45）个/10⁶细胞〕，但两者间的差异无统计学意义（t=1.252，P>0.05）。前者的SFC阳性率达到100%，而后者仅为50%。结论  通过优化ELISPOT方法中的细胞密度和刺激肽浓度，进一步提升了该法在重组痘苗病毒艾滋病疫苗细胞免疫评价中的灵敏度。

Objective  To optimize enzyme-linked immunospot assay (ELISPOT)  to improve its sensitivity in evaluation on the cellular immunity induced by recombinant Tiantan vaccinia virus (rTV)-based AIDS vaccine. Methods  BALB/c mice were intradermally injected with the above vaccine or a control vaccinia virus, 50 μl per mouse, and their spleens were sterilely collected after 6 weeks. The IFN-γ-secreting lymphocytes--spot-forming cells (SFCs) were detected by ELISPOT. The influence of different spleen lymphocyte densities (2×10⁵, 1×10⁶, 2×10⁶ cells/well) and stimulating peptide concentrations (2, 4 μg/ml) on SFC was compared. The comparison among multiple groups and between two groups was conducted by one-way ANOVA and t-test, respectively. Results There was no significant difference of SFC number among 3 cell density groups (F=0.023, P>0.05) and between 2 peptide concentration groups (t=-1.762-0.110, all P>0.05). When the cell density and peptide concentration were 2×105 cells/well and 4 μg/ml, respectively, the number of SFCs in vaccine group was (107.00±42.01) /10⁶ cells, higher than (76.55±64.45) /10⁶ cells obtained from previous experimental condition of 1×10⁶ cells/well cell density and 2 μg/ml peptide concentration, although no significant difference (t=1.252, P>0.05) was observed. The SFC positive rates were 100% and 50%, respectively. Conclusion   The sensitivity of ELISPOT in evaluation on the cellular immunity induced by rTV-based AIDS vaccine is further improved through optimizing cell density and stimulating peptide concentration.