摘要
目的制备抗流感嗜血杆菌外膜蛋白P6的单克隆抗体(mAb)并进行鉴定。方法以重组P6蛋白腹腔免疫BALB/c小鼠,常规细胞融合技术制备杂交瘤细胞株;间接ELISA筛选阳性细胞克隆并检测染色体数目;测定细胞培养上清的mAb效价及特异性,鉴定mAb的免疫球蛋白类、亚类及抗原结合表位。结果获得2株抗流感嗜血杆菌外膜蛋白P6的杂交瘤细胞株α2G3和γ2C4,染色体众数分别为103与95;间接ELISA结果证明细胞培养上清最高效价分别为1∶256和1∶512,未出现与其他种类细菌的交叉反应;α2G3为IgG2b、γ2C4为IgM,均为κ型;两者可能识别P6蛋白不同表位。结论成功制备了抗流感嗜血杆菌外膜蛋白P6的mAb。
Objective To prepare and identify monoclonal antibody against Haemophilus influenzae（ Hi） outer membrane protein P6. Methods Recombinant protein P6 as an immunogen was administered intraperitoneally to BALB /c mice. The splenocytes of the mouse were isolated from spleen and hybridized with Sp2 /0 myeloma cells. Indirect ELISA was used for screening hybridoma and the number of chromosomes in hybridoma cells was determined by karyotype analysis. The titers and specificity of monoclonal antibodies in their culture supernatant were detected by indirect ELISA. The immunoglobulin class,subclasses and type of the monoclonal antibody were identified with colloidal gold labeled IsoQuickTMstrips. Results Two hybridoma cell lines designated α2G3 and γ2C4 were obtained. Karyotype analysis showed that the chromosome numbers ofα2G3 and γ2C4 were 103 and 95,respectively. The highest titers of antibodies in their culture supernatant were 1∶256 and1∶512,respectively. Both monoclonal antibodies only reacted with standard or clinical isolated strains of Hi,and they both did not react with other bacteria. A2G3 was IgG2 b,and γ2C4 was IgM,both of which were kappa light chains. They could recognize different antigen epitope of protein P6. Conclusion Two hybridoma cell lines producing the monoclonal antibodies against protein P6 of Hi outer membrane are obtained.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第10期1051-1053,1061,共4页
Chinese Journal of Cellular and Molecular Immunology
关键词
流感嗜血杆菌
外膜蛋白P6
杂交瘤
单克隆抗体
Haemophilus influenzae
outer membrane protein p6
hybridoma
monoclonal antibody
