【摘要】 目的制备霍乱弧菌鞭毛蛋白单克隆抗体(mAb)、建立双抗体夹心ELISA并初步应用于食品检测。方法采用差速离心法提取霍乱弧菌Vc75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、稳定分泌抗霍乱弧菌鞭毛蛋白mAb的杂交瘤细胞株,命名为VcNo.1~VcNo.6,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VcNo.6抗体建立了霍乱弧菌双抗体夹心ELISA,该方法的检测灵敏度达到103CFU/mL菌液,通过采用100株霍乱弧菌和101株非霍乱弧菌进行特异性实验,100株霍乱弧菌呈阳性反应,101株非霍乱弧菌呈阴性反应,结果表明VcNo.6抗体具有良好的特异性。在基质添加试验中,最低检出限为1 CFU/g样品。结论成功制备了霍乱弧菌鞭毛蛋白mAb,并将其应用于双抗体夹心ELISA,该方法的检测灵敏度达到103CFU/mL菌液,与所测试的非霍乱弧菌没有交叉反应。更多还原

【Abstract】 Objective To prepare the monoclonal antibodies( mAbs) against flagellin core protein of Vibrio cholerae and establish the double-antibody sandwich ELISA method for testing Vibrio cholerae from food products. Methods BALB /c mice were immunized with flagellin extracted from Vibrio cholerae Vc75 by differential centrifugation. The splenocytes from the immunized mice were fused with Sp2 /0 myeloma cells when the antibody titer in serum reached 1∶32 000. The hybridoma cell lines were obtained by regular subcloning and used to generate ascites. And mAbs reacting to Vibrio cholerae flagellin were achieved by purified from the ascites. Results Six hybridoma cell lines stably secreting mAbs against Vibrio cholerae flagellin were taken and named VcNo. 1-VcNo. 6. The mAb titer in serum by indirect ELISA was 1∶2 × 106. SDS-PAGE showed that the flagellin protein molecular weight( M r) was 44 000 and the purity was high. Double-antibody sandwich ELISA method was set up using VcNo. 6 antibody for detecting Vibrio cholerae. The sensitivity reached 103CFU /mL. The ELISA method showed high specificity to Vibrio cholerae through testing 100 Vibrio cholerae( 100% positive) and 101 non-Vibrio cholerae strains( 100% negative). The detection limit was 1 CFU/g sample in artificial contaminated samples. Conclusion The mAbs against flagellin core protein of Vibrio cholerae was successfully prepared and used to set up the double-antibody sandwich ELISA. The mAb of VcNo. 6 was highly specific to Vibrio cholerae. The sensitivity of the established ELISA was as high as103CFU /mL. Moreover,it did not react to non-Vibrio cholerae strains. Therefore,the mAbs of VcNo. 6 could be widely used in Vibrio cholerae detection from food samples as well as clinical samples.更多还原