Establishment of DAS-ELISA Detection for Streptococcus agalaciate in Tilapia
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摘要: 通过杂交瘤技术制备抗罗非鱼无乳链球菌SIP融合蛋白单克隆抗体2株:1C9和6G5, 均为IgM, 效价分别为10-5和10-4, 间接ELISA结果显示单克隆抗体1C9对无乳链球菌具有良好的检测特异性和灵敏性;同时制备兔抗罗非鱼无乳链球菌SIP融合蛋白多克隆抗体及其HRP酶标记物, 兔抗SIP蛋白多克隆抗体的效价为10-6, 经标记的抗体效价为10-4, 最佳抗体工作浓度为1∶400。建立双抗体夹心ELISA (DAS-ELISA) 检测罗非鱼无乳链球菌的方法, 具良好的特异性, 检测灵敏度为0.85×106 CFU·mL-1。双抗体夹心ELISA检测实际应用结果与双重PCR检测比较, 相对符合率为98.33%, 检测结果可靠性高。Abstract: To develop a detection method for Streptococcus agalaciate in tilapia, a double-antibody sandwich ELISA (DAS-ELISA) was established using the SIP monoclonal antibody (McAb) as the capture antibody and the HRPlabelled rabbit polyclonal antibodies against SIP as the detecting antibody.Two hybridoma strains, 1C9 and 6G5, that secreted antibody against SIP of S.agalaciatein tilapia, were prepared and characterized.The titers of these two monoclonal antibodies could reach 1∶105 and 1∶104, and their isotypes were IgM.Indirect ELISA assay indicated that McAb 1C9 had a specific and sensitivity detection for S.agalaciate.Polyclonal antibodies against SIP were developed and conjugated with HRP.The titers of them were 1∶105and 1∶104.The optimal concentration of HRP-labelled antibody was 1∶400.The ELISA had no cross-reaction with S.iniae, S.suis or S.pluranimalium, and the minimum amount of S.agalaciate could be detected by this DAS-ELISA was 0.85×106 CFU·mL-1.The results indicated that the ELISA method was quick, sensitive, reproducible and applicable for the detection of S.agalaciatein tilapia.
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Key words:
- tilapia /
- Streptococcus agalaciate /
- SIP /
- McAb /
- DAS-ELISA
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