用竞争结合法检测HLAⅠ类分子抗原呈递能力

An useful method to analysis MHC class Ⅰ peptide binding

目的 建立弱酸处理后荧光素标记肽竞争结合法分析MHCⅠ类分子抗原呈递能力的方法 ,并评估其实用性。方法 pH2 .7～ 3.1的柠檬酸缓冲液对B淋巴母细胞进行短时处理后 ,即用IMDM培养液中和pH ,然后加入β2微球蛋白、荧光素标记的 9肽及竞争肽共同孵育 ,FACs检测细胞表面的平均荧光强度 ,以达到 5 0 %抑制所需的竞争肽浓度作为衡量竞争肽与MHC分子结合能力的指标。结果 通过不同pH的柠檬酸缓冲液处理、处理后不同时间加 β2微球蛋白和 /或荧光素标记肽、加入不同稀释度的竞争肽等角度 ,对方法进行评估 ,结果显示本方法操作简便、结果可靠、非特异性吸附小。结论 在排除其它MHCⅠ类分子的干扰后 ,本方法适合在国情条件下广泛开展MHCⅠ类分子抗原呈递能力及T细胞肽表位筛选等研究。

Objective To establish and evaluate a method of competition binding assay to MHC class Ⅰ molecules based on mild acid treatment described by van der Burg. Methods BLCL cells were treated with ice-cold citric acid-phosphat buffer (pH2.7-3.1) and buffered immediately using IMDM. After washing with IMDM, cells were incubated with β2m, fluorescein labeled reference peptide and competitor peptide. The mean fluorescence value was tested by a FACscan. Results β2m or FL-peptide were added after acid treatment with different time and pH, and a series end concentration of competitor peptide were incubated with FL-peptide. The results suggested that the method is easy to perform, sensitive and has low nonspecific adhering to other cell components. Conclusion The assay has a number of advantages compared to other method. The interference from other MHC class Ⅰ molecules was excluded. So the method can be used to investigate the peptide-MHC class Ⅰ binding and screen the T cell epitopes.