一种IFN-γ ELISPOT检测方法的建立  

Development of ELISPOT assay for IFN-γ

作　　者：刘培培 王洪芳 吕品 王少雄 

机构地区：[1]上海泽润生物科技有限公司分析制剂室,上海201203

出　　处：《中国免疫学杂志》2012年第1期62-66,共5页Chinese Journal of Immunology

摘　　要：目的:建立检测一种融合蛋白疫苗诱导特异性细胞免疫的ELISPOT实验方法,通过分析免疫小鼠分泌IFN-γ的水平,评价疫苗诱导的细胞免疫应答效应。方法:以融合蛋白疫苗免疫C57BL/6小鼠,14天后加强免疫一次,于第28天无菌取脾,分离脾淋巴细胞,计数并调整细胞浓度,在已包被好的ELISPOT板中分别加入细胞悬液和刺激肽进行刺激培养。裂解细胞,依次加入酶标二抗、底物、显色液,在ELISPOT读板仪进行斑点计数和分析。结果:ELISPOT检测结果与动物免疫途径、细胞培养时间、细胞密度、肽浓度等条件密切相关。实验结果证明:采用皮下注射的免疫途径,免疫效果最佳;小鼠脾淋巴细胞浓度为4×105个/孔,加入1μg/孔的刺激肽刺激培养24～48小时的条件下,产生的特异性斑点数最多,本底干扰小,有利于结果判断。结论:实验建立了IFN-γELISPOT检测方法,可用于该融合蛋白疫苗细胞免疫研究。Objective：To develop an ELISPOT assay for quantifying the IFN-γ secretion level and evaluating the fusion protein vaccine-induced cellular immune response. Methods：C57BL/6 mice were immunized subcutaneously with vaccine, a second equivalent dose was given 14 days later. Two weeks after the last booster,separated Spleen cells and adjusted cell concentration and added to the pre-cdated ELISPOT plate, co-cultured with stimulated peptide in 37 ℃ ,5 % CO2 for several hours. After lysing the splenocytes, the bi- otinylated anti-mouse IFN-γ monoclonal antibody, streptavidin and the substrates were added orderly. Finally, the colored spots were counted by ELISPOT Reader. Results： The specifications of IFN-γ ELISPOT assay were closely related to immunized route, stimulated peptide concentration, stimulation time, cell concentration, etc. The optimal immunized route was abdominal injection subcutaneously. When the assay was conducted with seeded Splenocytes concentration of 4 × 10^5/well, and stimulated with peptide of 1 μg/well for 24- 48 h,good specificity for spots counting and relatively low experimental error could be achieved. Conclusion：The developed IFN-γ ELISPOT assay can be used to evaluate the article mentioned fusion protein vaccine-induced cellular immune response successfully.

关 键 词：融合蛋白疫苗 细胞免疫 ELISPOT IFN-Γ 

分 类 号：R392[医药卫生—免疫学]