【摘要】 目的制备特异性和灵敏度较高的抗H5亚型禽流感病毒单克隆抗体,建立双抗体夹心ELISA方法,为进一步研究检测试剂盒提供基础。方法以H5亚型禽流感病毒为抗原,免疫BALB/c小鼠,通过细胞融合及杂交瘤细胞筛选,获取抗H5亚型禽流感病毒单克隆抗体,用Ig亚类ELISA试剂盒鉴定抗体亚类,间接ELISA法检测抗体效价及特异性。HRP标记单抗并建立双抗夹心ELISA检测方法,经方阵试验优化ELISA反应体系。结果获得配对的2株可分泌特异性单抗的阳性细胞株3C9、6F5,腹水抗体效价在1∶105以上,Ig G亚类均为Ig G2a,轻链为κ链。优化的双抗夹心检测体系能检测多种H5亚型禽流感病毒,与H9亚型和H7亚型均无交叉反应。结论成功获得高效价的抗H5亚型禽流感病毒单克隆抗体,建立抗H5亚型禽流感病毒单克隆抗体双抗体夹心ELISA检测方法。更多还原

【Abstract】 Objective: To make the preparation of specific and highly sensitive monoclonal antibodies against H5 subtype avian influenza virus( AIV-H5),which will be used in the establishment of a sandwich ELISA method and as a basic work to develop ELISA Kit. Methods: Balb/c mice were immunized with AIV-H5 and then positive clones of monoclonal antibody( McAb) were obtained after cell fusion and hybridoma selection. The ascites titers,the subclasses type,and the specificity of McAbs were tested by Ig subtype ELISA kit and ELISA,respectively. Moreover,optimization of the sandwich enzymelinked immunosorbent assay was studied by matrix assay. Results: Two of positive hybridomas were screened out,named 3 C9 and6 F5 whose ascites titers of McAbs were over 1∶ 105. It proved that the Ig subtypes of 3 C9 and 6 F5 McAbs were Ig G2 a,and the light chain was kappa. The DAS-ELISA could detect H5 AIV isolates,but not react with H9 and H7 isolates. Conclusion:Specific McAbs against H5 AIV are achieved and can be applied to the development of a double-antibody sandwich ELISA method.更多还原