摘要
目的制备抗细粒棘球绦虫Ediag A864单克隆抗体,并进行鉴定。方法以细粒棘球绦虫Ediag A864蛋白为抗原,经皮下多点免疫BALB/c小鼠,共免疫4次。末次免疫3 d后制备小鼠脾细胞,在聚乙二醇(PEG)的作用下与小鼠骨髓瘤细胞SP2/0进行细胞融合,间接ELISA法筛选出阳性杂交瘤细胞株进行克隆,检测其染色体数及分泌的单克隆抗体亚型。采用体内培养法大量制备抗细粒棘球绦虫Ediag A864单克隆抗体,并经正辛酸-饱和硫酸铵法和Hi Trap Protein G HP亲和层析结合法纯化。结果经筛选克隆共获得4株可产生细粒棘球绦虫Ediag A864单克隆抗体的杂交瘤细胞株:2D12、3H4、4A6和6E5,细胞染色体数分别为97、101、98和96,重链亚型均为Ig G1,轻链亚型均为Kappa。2D12、3H4腹水效价为2×105,4A6和6E5腹水效价为1×105。纯化后的2D12抗体可见相对分子质量为46 000和26 000的重链和轻链条带。结论本实验获得的2D12是一株稳定的杂交瘤细胞株,且可产生高效价细粒棘球绦虫Ediag A864单克隆抗体,为细粒棘球绦虫病的免疫诊断奠定了基础。
Objective To prepare and identify the monoclonal antibody（Mc Ab） against Ediag A864 of Echinococcus granulosus. Methods BALB / c mice were immunized s. c. with Ediag A864 of E. granulosus in several sites for 4 times,of which the spleen cells were collected 3 d after the last immunization and fused with SP2 / 0 myeloma cells by using PEG. Positive hybridoma cell strains were screened by indirect ELISA for cloning, of which the chromosome number and subtype of Mc Ab secreted were tested. The Mc Ab was prepared in a large quantity in culture in vivo, and purified by ncaprylic acid-saturated ammonium sulphate method and Hi Trap Protein G HP affinity chromatography. Results Four hybridoma cell strains secreting Mc Ab against Ediag A864 of E. granulosus were obtained and named as 2D12, 3H4, 4A6 and 6E5, of which the chromosome numbers were 97, 101, 98 and 96, respectively, while all the heavy chain subtypes of secreted Mc Abs were Ig G1, and the light chain subtypes were Kappa. The titers of Mc Abs secreted by strains 2D12 and3H4 were 2 × 10^5, while those by strains 4A6 and 6E5 were 1 × 10^5. The purified Mc Ab secreted by strain 2D12 consisted of a heavy and a light chains with relative molecular masses of 46 000 and 26 000 respectively. Conclusion The obtained 2D12 was a stable hybridoma cell strain secreting high titer Mc Ab against Ediag A864 of E. granulosus,which laid a foundation of immune diagnosis of infection with E. granulosus.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第6期606-609,共4页
Chinese Journal of Biologicals
基金
国家公益性行业(农业)科研专项(201303042)
