论文部分内容阅读
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 synthesize a monoclonal antibody against GPP of blood glycoprotein using synthetic peptide as an immunogen. Methods The synthetic peptide 17TKSYISSQTNGETGQLVHRF36 consisting of the 17th to the 36th amino acids of the GPB protein in the first glycoprotein of human glycophorin GPBs was used to immunize BALB / c mice with the synthetic peptide linked with keyhole limpet hemocyanin (KLH) carrier protein . Murine monoclonal antibody was prepared by hybridoma technology. After selective culture and sub-cloning by limiting dilution, positive hybridoma cell lines were screened by ELISA and healthy mice were inoculated intraperitoneally with positive hybridoma cells to prepare mouse ascites fluid, erythrocyte agglutination test, Specific erythrocytes, specific rare erythrocyte agglutination test, and Western-Blotting were used to identify the antibody specificity. The murine m Ab subclass was used to identify IgG subtype of antibody. Results One McAb positive to mAb was obtained by ELISA screening. The McAb was identified by murine m Ab subtype and the monoclonal antibodies were Ig G3 and Kappa type. The mouse ascites fluid and all the erythrocytes were agglutinated (including Ss, SS, ss, MM, MN, NN phenotypes) in both saline and anti-mouse globulin media without agglutination with the rare MkMk cells. The ascites fluid was reacted with O-type erythrocytes treated with papain, pineapple, fig and trypsin, and there was no significant change in agglutination strength and non-enzymatic treatment of erythrocytes. Western-Blotting hybridization showed that the hybridization band between mouse ascites fluid and erythrocyte membrane protein is located at the GPB. Conclusion The preparation of a hybridoma cell line secreting anti-GPB m Ab has laid the foundation for further experimental research.