多抗间接ELISA方法的建立及其在海洋细菌快速检测中的应用

Establishment of Polyclonal Antibody Indirect Enzyme-Linked Immunosorbent Assay (ELISA) and the Application in the Rapid Detection of Marine Bacteria

中文关键词: 间接ELISA  蜡样芽孢杆菌  溶藻胶弧菌  兔抗血清  对虾

英文关键词: Indirect ELISA  Bacillus cereus  Vibrio alginolyticus  Antisera  Shrimp

中文摘要:

      为了从对虾体内分离出的菌株中快速筛选出蜡样芽孢杆菌和溶藻胶弧菌，将蜡样芽孢杆菌与溶藻胶弧菌作为抗原，免疫SPF新西兰大白兔，获得免疫血清，效价均高于1 : 2000。将免疫兔血清作为一抗，HRP-羊抗兔血清作为二抗，建立了蜡样芽孢杆菌和溶藻胶弧菌快速检测的间接ELISA方法。此方法中，兔抗血清最佳稀释度为1 : 10000，菌液最佳包被浓度为106 CFU/ml；HRP-羊抗兔血清最佳稀释浓度为1 : 1000，可检测细菌最低浓度为104 CFU/ml。抗蜡样芽孢杆菌血清与苏云金芽孢杆菌有交叉反应，抗溶藻胶弧菌血清与其亲缘相近菌株无交叉反应，具有较强的特异性。2012年和2013年，分别从斑节对虾、中国对虾、凡纳滨对虾等9批样本中分离纯化到109株海洋细菌，利用建立的多抗间接ELISA方法对其中部分菌株进行快速检测，共检测出6株溶藻胶弧菌，未检测出蜡样芽孢杆菌。

英文摘要:

      In order to rapidly detect Bacillus cereus and Vibrio alginolyticus from shrimp, we immunized the specific pathogen free (SPF) of New Zealand white rabbits with V. alginolyticus and B. cereus and collected the antisera. The titer of the polyclonal antibody was higher than 1: 2000. The antisera were used as the primary antibody and the goat anti-rabbit IgG-HRP was used as the secondary antibody in indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of V. alginolyticus and B. cereus. The optimum dilution of the antiserum was determined to be 1:10000 in indirect ELISA. The optimum coated concentration of the antigen was determined to be 106 CFU/ml. The goat anti-rabbit IgG-HRP was determined to be 1: 1000. We tested the sensitivity of the serum and the lowest detectable concentration of the two strains was 106 CFU/ml. The anti-B. cereus serum had cross reaction only with B. thuringiensis. The cross reaction of the anti-V. alginolyticus serum with other bacterial species in the genus of Vibrio showed negative results. We purified 109 strains of marine bacteria from 9 sample batches including Penaeus monodon, Fenneropenaeus chinensis and Penaeus vanmamei, and these strains were timely identified using the established indirect ELISA method. Six strains of Vibrio were detected and no Bacillus was identified.