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目的制备副溶血性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副溶血性弧菌ATCC 17802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。多次克隆化培养后获得杂交瘤细胞株并制备腹水,纯化后得到抗体。结果获得6株持续、稳定分泌抗副溶血性弧菌鞭毛蛋白mAb的杂交瘤细胞株,命名为VpNo.1～VpNo.6,抗体效价高达1∶2×106。对抗体进行了Ig类和亚类检测以及Western blot法鉴定,其Ig亚类依次为IgG1、IgG1、IgM、IgG1、IgG2a和IgM。SDS-PAGE结果显示,提取出的鞭毛蛋白相对分子质量(Mr)为42 000并且纯度较高。采用VpNo.6抗体建立了副溶血性弧菌双抗夹心ELISA,该方法的检测灵敏度达到103CFU/mL菌液,通过采用134株副溶血性弧菌和74株非副溶血性弧菌进行特异性实验,结果表明134株副溶血性弧菌呈阳性反应,74株非副溶血性弧菌呈阴性反应,VpNo.6抗体具有非常好的特异性。在基质添加试验中,最低检出限为21 CFU/g样品。结论成功制备了副溶血性弧菌鞭毛蛋白mAb,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,与所测试的非副溶血性弧菌没有交叉反应。
Objective To prepare monoclonal antibodies against Vibrio parahaemolyticus flagellin (mAb) and to establish a double-antibody sandwich ELISA. Methods The flagellin of Vibrio parahaemolyticus strain ATCC 17802 was extracted by differential centrifugation and used as the antigen to immunize BALB / c mice. When the antiserum titer reached 1:32 000, Several Sp2 / 0 myeloma cells are fused. After multiple cloning and culture, the hybridoma cell line was obtained and the ascites was prepared. After purification, the antibody was obtained. Results Six hybridoma cell strains secreting anti-Vibrio parahaemolyticus flagellin mAb were obtained, named as VpNo.1 ~ VpNo.6. The antibody titers reached 1: 2 × 106. Antibodies were tested for Ig and subclasses and Western blot. The Ig subclasses were IgG1, IgG1, IgM, IgG1, IgG2a and IgM. SDS-PAGE results showed that the relative molecular mass (Mr) of the extracted flagellin was 42 000 and its purity was high. VpNo.6 antibody was used to establish a Vibrio parahaemolyticus double-antibody sandwich ELISA. The detection sensitivity of the method was 103CFU / mL. Through the use of 134 strains of Vibrio parahaemolyticus and 74 strains of non-Vibrio parahaemolyticus, The results showed that 134 strains of Vibrio parahaemolyticus were positive and 74 strains of non-Vibrio parahaemolyticus were negative. VpNo.6 antibody had very good specificity. In the matrix addition test, the minimum detection limit is 21 CFU / g sample. Conclusion Vibrio parahaemolyticus flagellin mAb was successfully prepared and applied to double-antibody sandwich ELISA. The detection sensitivity of the method was 1 × 103CFU / mL, and there was no cross reaction with the tested V. parahaemolyticus .