分泌抗春雷霉素单克隆杂交瘤细胞株的建立及免疫学特性鉴定

Establishment of Hybridoma Cell Lines Secreting Anti-Kasugamycin Monoclonal Antibody and Identification of Their Immunological Properties

【目的】制备高灵敏、高特异性的抗春雷霉素单克隆抗体,并对其免疫学特性进行鉴定。【方法】采用EDC法将KSM分别偶联于载体蛋白BSA和OVA上,分别合成免疫原KSM-BSA和包被原KSM-OVA,经SDS-PAGE鉴定后,免疫Balb/c小鼠,免疫间隔4周,4次免疫后,利用间接ELISA和间接竞争ELISA测定其多抗血清效价和敏感性,选出效价高、敏感性好的小鼠进行细胞融合,经过多次亚克隆筛选出分泌高敏感的特异性抗KSM单克隆抗体的杂交瘤细胞株,体内诱生腹水法制备抗KSM的单克隆抗体并对其免疫学特性进行鉴定。【结果】SDS-PAGE鉴定结果显示KSM-BSA成功偶联,间接ELISA检测显示免疫的6只小鼠血清效价均达1:104以上,其中3号小鼠多抗血清敏感性最好,半数抑制浓度IC50为73.9ng.mL-1,融合后筛选出两株杂交瘤细胞株1G7和2C8,亚型均为IgG1型,其细胞上清效价分别为1:5.12×103和1:1.28×103,腹水效价分别为1:5.12×105和1:2.56×105,抗KSM的单克隆抗体对春雷霉素的IC50为8.902ng.mL-1,与其它竞争物的交叉率均小于0.03%。【结论】本试验成功合成了春雷霉素人工抗原,并制备了敏感性高、特异性好的单克隆抗体,为春雷霉素免疫学快速检测方法的建立奠定了基础。

[Objective] The aim of the study is to generate high sensitivity and high specificity monoclonal antibody against kasugamycin and identify its immunological characteristics. [ Method] KSM was coupled with the carrier protein BSA and OVA by using EDC method and the immunogen KSM-BSA and coating antigen KSM-OVA were synthesized respectively, and SDS-PAGE was used to identify KSM-BSA. Balb/c mouse was immunized by using KSM-BSA. Immunization interval was 4 weeks. The titer ofpolyclonal antibody was detected by indirect ELISA and blocking ELISA after four times immunization. The high titer and high sensitivity mouse was selected for cell fusion. High sensitivity and high specificity monoclonal antibody was prepared after several times subcloning,and the immunological characteristics were characterized. The preparation of ascites was carried out using in vivo induction method. [Result] SDS-PAGE, results showed that KSM-BSA artificial antigen was synthesized successfully.Indirect ELISA showed a high titer above 1：104 of the antiserum of all the six Balb/c mouse. The sensibility of antiserum of No.3 mice was the best, which ICs0 was 73.9ng＇mL-l. Hybridoma cell lines of 1G7 and 2C8 were screened after cell fusion, all the isotypes of the mAb were IgG1.Titers of the mAb were 1：5.12x103 and 1：1.28x103 in supernatant, l：5.12xl05 and 1：2.56~105 in ascites. The ICs0 of KSMmAb was 8.902 ng＇mLx to kasugamycin. The rate of cross reaction of the mAb with other compounds was less than 0.03%. [Conclusion] KSM artificial antigens were synthesized successfully and the monoclonal antibody with high sensitivity and specificity was obtained. This study has laid a foundation for a KSM immunological fast detection method.