创伤弧菌鞭毛蛋白单克隆抗体的制备及在食品检测中的应用

发布时间：2018-06-07 23:47

本文选题：创伤弧菌 + 鞭毛蛋白　；参考：《细胞与分子免疫学杂志》2013年10期

【摘要】：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。
[Abstract]:Objective to prepare the monoclonal antibody (mAb) of Vibrio vulnificus flagellin (mAb) and establish a double anti sandwich ELISA. method to extract flagellin of Vibrio vulnificus ATCC 1.1758 by differential centrifugation, and to immunize BALB/c mice with antigen, and when the antiserum titer reached 1: 32000, the splenocytes were taken with the logarithmic Sp2/0 myeloma cells with good growth. Hybridoma technique and ELISA were used to prepare and screen hybridoma cell lines secreting mAb. A finite dilution method was used to culture positive kork. The ascites were prepared and purified to obtain antibodies. The results obtained 5 hybridoma cell lines that secreted the flagellin mAb of Vibrio vulnificus steadily and were named VVNo.1~VVNo.5. The antibody titer was up to 1: (2 * 106). .SDS-PAGE results showed that the relative molecular mass (M R) of the extracted flagellin was 44000 and the purity was high. Using VVNo.5 antibody, the double anti sandwich ELISA of Vibrio vulnificus was established. The detection sensitivity of this method reached 103CFU/mL liquid. The specific experiment was carried out by using 12 strains of Vibrio vulnificus and 130 strains of Vibrio vulnificus, and 12 strains of Vibrio vulnificus were presented. The positive reaction, 130 non traumatic Vibrio showed negative reaction, the results showed that the VVNo.5 antibody had good specificity. In the matrix addition test, the minimum detection limit was 2 CFU/25 G by adding bacteria. Conclusion the monoclonal antibody of Vibrio vulnificus flagellin was successfully prepared, and it should be used in the double resistance sandwich ELISA. The sensitivity of the method was reached. Up to 1 x 103CFU/mL, there was no cross reaction with the tested non target bacteria.
【作者单位】：北京出入境检验检疫局;北京农学院;河北出入境检验检疫局;北京大学;
【基金】：国家质检总局公益性行业科研专项(201110034)
【分类号】：R392.11

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