IL-31单克隆抗体的制备及ELISA检测方法的建立  

Preparation of monoclonal antibodies against IL-31 and development of sandwich ELISA for IL-31

作　　者：董革辉[1] 魏培[2] 黄俊琼[3] 杜青波[3] 岳欢[3] 杜文胜[3] 谭艳芳[3] 孙万邦[2] 

机构地区：[1]遵义医学院第三附属医院骨科,遵义563000 [2]遵义医学院珠海校区免疫学教研室,珠海519041 [3]遵义医学院附属医院检验科,遵义563003

出　　处：《中国免疫学杂志》2014年第4期495-498,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(No.81260266);贵州省科技攻关项目(No.黔科合NY20063039号);贵州省优秀科技教育人才省长专项资金项目(No.黔省专合200951号)

摘　　要：目的:制备抗人IL-31单克隆抗体,建立ELISA检测体系。方法:采用合成免疫原免疫BALB/c小鼠,通过杂交瘤技术制备抗IL-31单克隆抗体,用HRP标记单克隆抗体,ELISA和Western blot法检测抗体的亚类、滴度和特异性;采用ELISA技术制备IL-31检测试剂盒,并对自制试剂盒进行评价。结果:获得了两株稳定产生抗IL-31单克隆抗体的杂交瘤细胞,抗体亚类均为IgG3,抗体特异性好,杂交瘤细胞诱生的腹水抗体效价达106。试剂盒检测灵敏度为5.726 pg/ml,检测IL-1、IL-6、TNF-α、RF的P/N值均小于2.1,为阴性,靶抗原IL-31的检测结果为阳性(P/N值=13.76);自制试剂盒检测特应性皮炎血清IL-31水平为(532.83±90.39)pg/ml,对照组血清IL-31水平为(103.31±19.17)pg/ml,皮炎组明显高于对照组,有显著性差异(P<0.001)。结论:成功制备了抗IL-31单克隆抗体,建立的ELISA试剂盒可用于临床检测IL-31。Objective：To prepare monoclonal antibodies against IL-31 and develop an ELISA kit for IL-31. Methods： Hybri- doma lines were obtained by fusing myeloma and spleen cells from BALB/e mice immunized with synthetic antigen BSA-IL-31. The mAbs from mice injected with hybridoma lines were purified and conjugated to HRP. The subtype, titer and specificity of monoclonal antibodies were determined by ELISA and western blot. Based on epitope of IL-31 and IL-31 mAbs HRP-conjugation, the sandwich ELISA kit for IL-31 was developed. Results： Two clones derived hybridoma （8A1 and 8C3） were anti-IL-31 producers with higher an- tibody secretion. Both of them were producing antibodies from IgG3 subclass, with titer of 106. The ELISA kit had an analytical sensi- tivity value of 5. 726 pg,/ml, however, no cross-reaction was detected in IL-1, IL-6, TNF-α and RF （ P/N 〈2.1 ）. The serum level of IL-31 in serum of AD patients was （532.83 ±90.39）pg/ml, which is significantly higher than those in healthy group. Conclusion： Specific monoclonal antibodies against IL-31 were produced and incorporated into the ELISA kit which can be used for detection of IL- 31 in serum samples.

关 键 词：IL-31 单克隆抗体 

分 类 号：R392-33[医药卫生—免疫学]