炭疽AVA疫苗特异性抗体分泌细胞检测方法的建立  

Development of a method for detecting AVA-antigen specific antibody secreting cells

作　　者：吕进[1] 张松乐[1] 董梅[1] 何蕊[1] 江海燕[1] 张良艳[1] 邢丽[1] 杨海荣[1] 王希良[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《免疫学杂志》2007年第4期452-455,共4页Immunological Journal

基　　金：国家"973"课题(2002CB513208)

摘　　要：目的 应用酶联免疫斑点（ELISPOT）技术,建立一种检测炭疽AVA免疫后特异性抗体分泌细胞的方法,用于检测和评价炭疽无菌培养滤液抗原特异性抗体形成细胞的功能.方法 无菌分离小鼠脾细胞,建立一系列特异性ELISPOT检测炭疽无菌培养滤液抗原特异性抗体分泌细胞的参数：包括抗原最佳包被浓度、细胞最适孵育时间、反应体系中细胞浓度以及亲和素抗体工作浓度的确定,同时检测上清中抗体效价,并以不相关抗原包被,检验方法的敏感性和特异性.结果 当抗原包被浓度为7.5μg/mL时,不同细胞浓度反应曲线最稳定 细胞数为5×10^6/mL时,不同包被浓度形成的斑点数适量,易于计数 且在抗体稀释度相同情况下,脾细胞孵育时间为3～6 h所获得的斑点数最清晰,背景着色最浅,容易被仪器识别 同时,特异性抗原包被组所获得的斑点频数高于无关抗原包被组（P＜0.01） 反应体系上清中未检测到抗原特异性抗体.结论 该方法敏感、特异,可用于AVA抗原免疫后体液免疫评价.Objective To establish a method for detecting AVA-antigen specific antibody-secreting cells （ASCs） with enzyme linked immunospot essay, which can be used to detect and evaluate the function of AVA-antigen specific ASCs, and to observe the humoral immune responses in mice after being inoculated with AVA. Methods The level of Ig and its subtype in sennn was detected. Mice were sacrificed and spleen cells were isolated. A series of parameters for detecting AVA specific ASCs was established： including the optimal concentration of coated antigen, optimal incubation time of spleen cells, and the performance concentration of Biotin-Igs. The sensitivity and specificity of the method was checked by detecting lgG levels in the culture supernatant and coating with irrelevant antigen. Results When antigen concentration was 7.5μg/mL, the reaction curve was the most stable compared with the others. The amount of spots in cell with concentration of 5× 10^6 mL^-1 were in moderate level and easy to be recognized. Besides, the detected spots were the clearest and the background was the lightest when the incubation time was 3 - 6 h comparing with the others under the same circmnstance. Lastly, the amount of spots coated with AVA-antigen was much higher than that coated with irrelevant antigen （ P 〈 0.01 ）. Antigen-specific ASC was undetected in the culture supernant. Comclusion The ELISPOT essays represent a sensitive, specific, and reliable tool for tracking humoral immune responses after AVA vaccination.

关 键 词：AVA(炭疽无菌培养滤液佐剂吸附苗) ELISPOT(酶联免疫斑点) ASC(抗体分泌细胞) SFC(斑点形成细胞) 

分 类 号：R446.6[医药卫生—诊断学]