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目的制备创伤弧菌鞭毛蛋白单克隆抗体(mAb)并建立双抗夹心ELISA。方法采用差速离心法提取创伤弧菌ATCC 1.1758菌株的鞭毛蛋白,并以此为抗原免疫BALB/c小鼠,抗血清效价达到1∶32 000时,取脾细胞与生长良好的对数期Sp2/0骨髓瘤细胞进行融合。采用杂交瘤技术和ELISA制备并筛选分泌mAb的杂交瘤细胞株,有限稀释法对阳性孔克隆化培养。制备腹水,纯化以得到抗体。结果获得5株持续、稳定分泌抗创伤弧菌鞭毛蛋白mAb的杂交瘤细胞株,命名为VVNo.1~VVNo.5,抗体效价高达1∶(2×106)。SDS-PAGE结果显示,提取出的鞭毛蛋白相对分子质量(M r)为44 000并且纯度较高。采用VVNo.5抗体建立了创伤弧菌双抗夹心ELISA,该方法的检测灵敏度达到103CFU/mL菌液,通过采用12株创伤弧菌和130株非创伤弧菌进行特异性实验,12株创伤弧菌呈阳性反应,130株非创伤弧菌呈阴性反应,结果表明VVNo.5抗体具有良好的特异性。在基质添加试验中,通过增菌,最低检出限为2 CFU/25 g样品。结论成功制备了创伤弧菌鞭毛蛋白单克隆抗体,并将其应用于双抗夹心ELISA,该方法的检测灵敏度达到1×103CFU/mL菌液,与所测试的非目标菌没有交叉反应。
Objective To prepare monoclonal antibodies against Vibrio vulnificus flagellin (mAb) and to establish a double-antibody sandwich ELISA. Methods The flagellar protein of Vibrio vulnificus ATCC 1.1758 was extracted by differential centrifugation and used as antigen to immunize BALB / c mice. When the antiserum titer reached 1:32 000, the spleen cells were incubated with well-logarithmic phase Sp2 / 0 myeloma cells. The hybridoma cell lines secreting mAbs were prepared and screened by hybridoma technique and ELISA, and the positive clones were cultured by limiting dilution method. Ascites is prepared and purified to give the antibody. Results Five hybridoma cell lines secreting Vibrio vulnificus Vibrio flagellin mAb were obtained and named as VVNo.1 ~ VVNo.5. The antibody titers reached 1: (2 × 106). The results of SDS-PAGE showed that the relative molecular mass (M r) of extracted flagellin was 44 000 and its purity was high. VVNo.5 antibody was used to establish Vibrio parahaemolyticus double antibody sandwich ELISA. The detection sensitivity of the method was 103 CFU / mL. By using 12 strains of Vibrio vulnificus and 130 strains of non-Vibrio vulnificus, The results showed that VVNo.5 antibody had a good specificity. In the matrix addition test, the minimum detection limit is 2 CFU / 25 g by enrichment. Conclusion The McAb against Vibrio vulnificus was successfully prepared and applied to double-antibody sandwich ELISA. The sensitivity of the method was 1 × 103CFU / mL, which was not cross-reactive with the non-target bacteria tested.