弧菌融合蛋白TDH-VVC-VMHA双抗体夹心ELISA检测-中国农学通报.PDFVIP

中国农学通报 2011,27(32):34-37 Chinese Agricultural Science Bulletin 弧菌融合蛋白TDH-VVC-VMHA 双抗体夹心 ELISA 检测方法的建立 刘秀萍，王铁良，唐 峰 （辽宁医学院畜牧兽医学院，辽宁锦州121001） 摘 要：为提高食品中副溶血弧菌、创伤弧菌和拟态弧菌毒素初筛的速度与效率，建立了双抗体夹心 ELISA 检测方法。以融合蛋白TDH-VVC-VMHA 包涵体为抗原免疫豚鼠，采用豚鼠抗融合蛋白免疫血 清为包被抗体，辣根过氧化物酶(HRP)标记羊抗豚鼠IgG 为标记抗体，应用棋盘滴定法确定ELISA 的最 适条件。结果表明，所建立的方法与副溶血弧菌、创伤弧菌、拟态弧菌的培养物上清有较明显的阳性反 应，与绿脓杆菌、金黄色葡菌球菌、李氏杆菌、甲型副伤寒沙门氏菌、猪霍乱沙门氏菌等5 种非弧菌属食 物中毒菌培养物上清未见交叉反应，与溶藻弧菌、河流弧菌和麦氏弧菌等3 种弧菌培养物上清也未见交 叉反应。这说明，该方法可以用于食品中副溶血弧菌、创伤弧菌和拟态弧菌毒素的快速同步免疫学检 测。 关键词：弧菌；融合蛋白；双抗体夹心ELISA 中图分类号：S851.4+3 文献标志码：A 论文编号：2011-1691 Development of Double Antibody Sandwich ELISA for Vibrio TDH-VVC-VMHA Fusion Protein Liu Xiuping, Wang Tieliang, Tang Feng （Institute of Animal Husbandry and Veterinary Medicine, Liaoning Medical University, Jinzhou Liaoning 121001） Abstract: In order to improve the detection speed and efficiency, a double antibody sandwich ELISA was developed to detect Vibrio parahaemolyticus，Vibrio vulnificus or Vibrio minicus in food simultaneously. The cytorrhyctes of the fusion protein were purified by the electroeluting to immunize cavia cobaya, anti-serum were uesed as capture antibody,goat anti-guinea pig IgG/HRP were uesed as labelled-antibody, optimizing condition was determined by use of the chessboard titration method. The results showed that, the double antibody sandwich ELISA having obvious positive reaction with the culture supernatant of Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, but no positive reaction with 8 other culture supernatant of contrast strains including Pseudom