Zhang Lei, Zhang Ximeng, Zhang Haiyu, Wei Haiyan, Ma Dan, Liu Xuesong, Cao Dong, Zeng Jing
Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Jul;29(7):734-8.
To prepare monoclonal antibodies (mAbs) against flagellin core protein of Vibrio (V.) parahaemolyticus and establish the double-sandwich ELISA for testing V.parahaemolyticus from food products.
BALB/c mice were immunized with flagellin which was extracted from V.parahaemolyticus ATCC17802 through differential centrifugation. The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells in log-phase growth when the antibody titer in serum was 1:32 000. The hybridoma cell lines were screened by regular subcloning approach and used to generate ascites. And mAbs reacting to V.parahaemolyticus flagellin were obtained by purifying from the ascites.
Six hybridoma cell lines secreting mAbs against V.parahaemolyticus flagellin were prepared and named VpNo.1-VpNo.6. The mAb titer in serum by indirect ELISA was 1:2×10(6);. The mAbs were subjected to Ig classes and subclasses detection and Western blotting. The Ig subclasses of these mAbs were IgG1, IgG1, IgM, IgG1, IgG2a and IgM, respectively. SDS-PAGE showed that the flagellin protein molecular weight (Mr;) was 42 000. Using VpNo.6, Double-sandwich ELISA kit was set up and applied in detecting V.parahaemolyticus, and its sensitivity was 10(3); CFU/mL. The ELISA showed VpNo.6 had a desirable specificity to V.parahaemolyticus in testing 134 V.parahaemolyticus and 74 non-V.parahaemolyticus strains. The lowest limit of detection was 21 CFU/g sample in the base-material addition test.
The monoclonal antibodies against flagellin core protein of V.parahaemolyticus were successfully prepared. The double-sandwich ELISA we established using VpNo.6 mAb had a sensitivity as high as 10(3); CFU/mL. The monoclonal antibody of VpNo.6 was highly specific to V.parahaemolyticus.
制备抗副溶血性弧菌鞭毛蛋白核心蛋白的单克隆抗体，并建立用于检测食品中副溶血性弧菌的双夹心ELISA方法。
采用差速离心法从副溶血性弧菌ATCC17802中提取鞭毛蛋白，免疫BALB/c小鼠。当血清抗体效价达到1:32 000时，取免疫小鼠的脾细胞与处于对数生长期的Sp2/0骨髓瘤细胞进行融合。采用常规亚克隆方法筛选杂交瘤细胞系并制备腹水。从腹水中纯化获得抗副溶血性弧菌鞭毛蛋白的单克隆抗体。
制备了6株分泌抗副溶血性弧菌鞭毛蛋白单克隆抗体的杂交瘤细胞系，命名为VpNo.1 - VpNo.6。间接ELISA法检测血清中单克隆抗体效价为1:2×10(6)。对单克隆抗体进行Ig类别和亚类检测及Western印迹分析。这些单克隆抗体的Ig亚类分别为IgG1、IgG1、IgM、IgG1、IgG2a和IgM。SDS - PAGE显示鞭毛蛋白分子量（Mr）为42 000。以VpNo.6建立双夹心ELISA试剂盒并应用于副溶血性弧菌检测，其灵敏度为10(3) CFU/mL。在检测134株副溶血性弧菌和74株非副溶血性弧菌菌株时，ELISA结果显示VpNo.6对副溶血性弧菌具有良好的特异性。基础材料添加试验中最低检测限为21 CFU/g样品。
成功制备了抗副溶血性弧菌鞭毛蛋白核心蛋白的单克隆抗体。用VpNo.6单克隆抗体建立的双夹心ELISA灵敏度高达10(3) CFU/mL，VpNo.6单克隆抗体对副溶血性弧菌具有高度特异性。
