时间分辨荧光免疫法检测日本鳗鲡的产酸克雷伯氏菌  

Time-Resolved Fluoroimmunoassay for Detecting Klebsiella Oxytoca in Anguilla Janponica

作　　者：卢洁[1] 郭松林[1] 冯建军[1] 王艺磊[1] 朱洪泉[1] 林鹏[1] 

机构地区：[1]集美大学水产学院,农业部东海海水健康养殖重点实验室,水产科学技术与食品安全省高校重点实验室,厦门361021

出　　处：《分析化学》2012年第11期1709-1714,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.30972281;31001136);福建省高等学校新世纪优秀人才支持计划(闽教科[2007]20号);集美大学创新团队基金项目(No.2010A001)资助

摘　　要：从皮肤点状出血的日本鳗鲡中分离出产酸克雷伯氏菌菌株B12,经福尔马林灭活后,注射新西兰兔,制备免疫抗血清,G蛋白亲和层析纯化得到抗体(IgG),以固定于微孔板的IgG作为捕获抗体,Eu^(3+)螯合物标记的IgG作为检测抗体,建立了产酸克雷伯氏菌的夹心型时间分辨荧光免疫检测法(TRFIA),并研究了抗体浓度、免疫时间及离解时间对检测的影响。本方法检出限为5.0×10~3 cfu/mL;线性范围5.0×10~3～1.0×10~7 cfu/mL,相关系数达0.99以上。交叉反应表明其具有较高的特异性,板内和板间相对标准偏差分别为5.64%和2.27%。将本方法标准化后,检测了29份人工感染的日本鳗鲡样品,包括鳃、肾、肠、肝和肌肉组织,结果满意。Klebsiella Oxytoca B12,isolated from diseased Anguilla janponica,was used for rabbit anti-serum preparation by injecting the formalin-inactivated bacteria.The rabbit immunoglobulin （IgG） was purified by SPA affinity column.A microplate sandwich immunoassay for the detection of K.Oxytoca was developed utilizing time-resolved fluoroimmunoassay（TRFIA）.Antibodies immobilized on microtiter plates were used to capture the bacteria.The bound antigen-antibody complexes were then detected using the same antibodies labeled with Eu^（3-） chelates.The effects of antibody concentration,imtnunoreaction time and dissociation time on assay were studied during assay development.The limit of detection was 5.0×10~3 cfu/mL and the linear range was 5.0×10~3—1.0×10~7 cfu/mL with the correlation coefficients more than 0.99.Cross reaction showed that it had high specificity.The intra-assay and inter-assay standard deviation were 5.6%and 2.3%,respectively. The method was optimized to detect the tissues including gill,kidney,intestine,liver and muscle from infected K.Oxytoca with satisfactory results.

关 键 词：时间分辨荧光免疫检测 产酸克雷伯氏菌 日本鳗鲡 

分 类 号：R446.6[医药卫生—诊断学]