Pfs25蛋白单克隆抗体的制备及双抗体夹心ELISA的建立  

Preparation of monoclonal antibody against Pfs25 protein and establishment of double antibody sandwich ELISA method

作　　者：李萍[1] 马亚茹[1] 陆俭[1] 雷清[1] 陈勇[1] 蒋琳[1] 

机构地区：[1]国药集团兰州生物制品研究所,甘肃兰州730046

出　　处：《细胞与分子免疫学杂志》2011年第12期1330-1334,共5页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的:制备恶性疟原虫子孢子囊表面膜蛋白Pfs25的单克隆抗体(mAb),建立检测Pfs25蛋白的双抗体夹心ELISA方法。方法:纯化毕赤酵母表达的重组Pfs25蛋白,并免疫BALB/c小鼠,采用骨髓瘤细胞Sp2/0与免疫BALB/c鼠脾细胞杂交的细胞融合技术,通过间接ELISA检测获得分泌抗Pfs25抗体的阳性杂交瘤细胞株,通过免疫F1鼠诱生腹水,纯化腹水,并进行mAb的各项生物学鉴定。辣根过氧化物酶(HRP)标记纯化后的抗体,以4B7为包被抗体,1B4为酶标抗体,建立了双抗体夹心ELISA法。结果:获得3株抗Pfs25的杂交瘤细胞株,其中2株有良好的稳定性和特异性。并建立了双抗体夹心ELISA检测法,检测有效范围在0.07～1 mg/mL,其检测灵敏度为41.6 ng/mL。结论:成功制备抗Pfs25蛋白的单克隆抗体,并建立了一种可用于Pfs25蛋白检测的双抗体夹心ELISA法,为Pfs25蛋白制备传播阻断型疟疾疫苗奠定了基础。AIM： To prepare the monoclonal antibody （mAb）against Pfs25 protein of Plasmodium falciparum, and establish the method of sandwich ELISA for detecting the Pfs25 protein. METHODS： Pfs25 protein the recombinant expressed by Pichia pastoris was purified. The purified Pfs25 protein as the antigen was used to immune the BALB/c mice, The secreting specific mAb positive cell strains, which were prepared by hybridizing the Sp2/0 myeloma cell and the spleen cell from immunized mice, were detected by indirect ELISA method. The ascites of mAb were collected from immunization F1 mice, and their biological properties were identified by indirect ELISA. The anti-Pfs25 antibody was la- beled by Horseradish Peroxidase （HRP）, the sandwich ELISA method to detect Pfs25 protein was established based on the anti-Pfs25 mAb 4B7 and 1B4 as coating and enzyme antibody, respectively. RESULTS： Three hybrido- ma cell lines secreting mAb against Pfs25 protein have been selected from the antibody positive hybridizing cells. The two of them have a better stability and specificity. The sandwich ELISA method detecting Pfs25 protein was established. Its detecte range was 0.07 - 1 mg/mL, and its sensitivity was 41.6 ng/mL. CONCLUSION： The anti-Pfs25 mAb are successfully prepared and the double antibody sandwich ELISA method detecting Pfs25 protein is established. Our study lay a foundation of developing transmission-blocking malaria vaccine with Pfs25 protein as antigen.

关 键 词：Pfs25蛋白 单克隆抗体 双抗体夹心ELISA 传播阻断型疟疾疫苗 

分 类 号：R392.11[医药卫生—免疫学]