用合成肽制备鼠源性抗-GPB单克隆抗体及特性鉴定

Preparation and characterization of a murine monoclonal antibody against human glycophorin B with a peptide

目的用合成肽作为免疫原,制备针对血型糖蛋白GPB的单克隆抗体细胞株。方法合成人血型糖蛋白GPBs上1段由GPB蛋白第17-36位氨基酸构成的肽段17TKSYISSQTNGETGQLVHRF36,用钥孔虫血蓝(KLH,keyhole limpet hemocyanin)载体蛋白联接的合成多肽免疫BALB/c小鼠。采用杂交瘤技术制备鼠源单克隆抗体,经选择培养和有限稀释亚克隆后,ELISA筛选阳性杂交瘤细胞株,健康小鼠腹腔接种阳性杂交瘤细胞,制备小鼠腹水,谱红细胞凝集试验、酶处理红细胞、特定稀有血型红细胞凝集试验,以及蛋白免疫印记(Western-Blotting)等技术鉴定抗体特异性;鼠源m Ab亚类试剂盒鉴定抗体Ig G亚型。结果 ELISA筛选获得阳性单抗细胞株1株,经鼠源m Ab亚类试剂盒鉴定,所制备的单克隆抗体属于Ig G3,Kappa型轻链。其小鼠腹水制备液与所有谱红细胞在盐水和抗鼠球蛋白介质中均产生凝集(包括Ss,SS,ss,MM,MN,NN表型),与稀有的MkMk细胞不发生凝集。腹水制备液与经木瓜酶、菠萝酶、无花果酶和胰蛋白酶处理的O型红细胞反应,凝集强度与非酶处理红细胞无明显改变。Western-Blotting杂交试验显示小鼠腹水稀释液与红细胞膜蛋白的杂交条带位于GPB的位置。结论制备1株可分泌抗-GPB m Ab的杂交瘤细胞株,为进一步的实验研究奠定了基础。

Objective To develop and identify monoclonal antibody（ m Ab） cell lines against human glycophorin B.Methods Monoclonal antibodies were prepared by hybridoma technique. A KLH-conjugated synthetic peptide corresponding to the GPBs sequence was used as immunogen. The specificity of m Ab was identified by indirect ELISA,hemagglutination tests reacted with panel reagent red blood cells,rare phenotype RBCs and various enzyme-treated RBCs. In addition,the m Ab ascitic fluids were analyzed by western blotting. Results One mouse monoclonal antibody against human red blood cell glycophorin B has been produced which belonged to Ig G3 subclass,Kappa type light chain. The m Ab ascitic fluids can react with protease-resistant determinant on GPB of RBC,but can not hemagglutinate with the rare Mk Mk cells. Western-Blotting results suggested that the m Ab recognized GPB bands. Conclusion One hybridoma cell secreting m Ab against high frequency antigen on GPB has been established which lays the foundation for future study.