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【目的】制备高灵敏、高特异性的百菌清(CTN)单克隆抗体,并对其免疫学特性进行鉴定。【方法】将CTN进行化学修饰后引入羟基活性基团,合成具有半抗原结构特征的百菌清衍生物(CTN-OH);采用N,N-羰基二咪唑法(CDI)将CTN-OH与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联合成人工免疫抗原CTN-BSA和包被抗原CTN-OVA,用紫外分光光度法(UV)和凝胶电泳(SDS-PAGE)进行鉴定后,免疫BALB/c小鼠,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CTN单克隆抗体的杂交瘤细胞株,体内诱生腹水法制备抗CTN的单克隆抗体并对其免疫学特性进行鉴定。【结果】UV和SDS-PAGE鉴定结果显示,CTN-BSA成功偶联,间接ELISA检测显示免疫的3只小鼠效价均达1﹕104以上,其中2号小鼠多抗血清敏感性最好,半数抑制浓度IC50为93.593 ng.mL-1,融合后筛选出4株杂交瘤细胞株1E8、1F4、2A10和2E2,其细胞上清效价分别为1﹕1.6×103、1﹕6×102、1﹕6×102、1﹕6×102,1E8株腹水效价为1﹕5.12×105,抗CTN单克隆抗体对CTN的IC50为21.6685 ng.mL-1,且具有较好的特异性。【结论】本试验成功合成了CTN人工抗原,并制备了敏感性高、特异性好的单克隆抗体,为CTN免疫学快速检测方法的建立奠定了基础。
【Objective】 To prepare highly sensitive and highly specific CTN monoclonal antibodies and to identify their immunological properties. 【Method】 CTN was chemically modified and introduced into hydroxyl reactive groups to synthesize chlorothalonil (CTN-OH) with the structural features of hapten. CTN-OH and CTN-OH were detected by using N, N-carbonyldiimidazole Bovine serum albumin (BSA) and chicken ovalbumin (OVA) conjugate synthetic immunogens CTN-BSA and coated antigen CTN-OVA, UV spectrophotometry (UV) and gel electrophoresis (SDS-PAGE) After identification, the BALB / c mice were immunized, the multi-antiserum titer and sensitivity were determined by indirect ELISA and indirect competitive ELISA, and the mice with high titer and sensitivity were selected for cell fusion. After several subclone screening A monoclonal antibody against CTN was prepared by in vitro inoculation of a hybridoma cell line secreting highly sensitive and specific anti-CTN monoclonal antibodies and its immunological properties were identified. 【Results】 The results of UV and SDS-PAGE showed that the CTN-BSA was successfully coupled. The indirect immunofluorescence assay showed that the titer of 3 immunized mice reached more than 1:10, among which the antiserum of No. 2 mouse was the most sensitive , The IC50 of half inhibitory concentration was 93.593 ng.mL-1. After hybridization, 4 hybridoma cell lines, 1E8, 1F4, 2A10 and 2E2, were screened and their cell supernatant titers were 1: 1.6 × 103 and 1: 6 × 102 , 1: 6 × 102, 1: 6 × 102, 1E8 strains of ascites titer of 1: 5.12 × 105, anti-CTN monoclonal antibody CTN IC50 of 21.6685 ng.mL-1, and has good specificity. 【Conclusion】 CTN artificial antigens were successfully synthesized in this study and monoclonal antibodies with high sensitivity and specificity were prepared, which laid the foundation for the establishment of rapid CTN immunodetection assay.