免疫PCR检测微量H5亚型禽流感病毒

Immuno-PCR Assay for Detection of H5 Hemagglutinin Subtype Avian Influenza Virus

为了有效检测低浓度禽流感病毒,笔者通过将高特异性的抗原-抗体反应与高灵敏性的PCR扩增技术相结合,建立了一种快速检测痕量H5亚型禽流感病毒的间接免疫PCR方法和间接"三明治"抗体夹心免疫PCR方法。以TopYield 96孔板为固相免疫吸附载体,以禽流感病毒H5蛋白为检测对象,通过一系列免疫反应及亲和素-生物素桥联作用,将生物素标记的报告DNA分子和生物素标记的禽流感病毒H5亚型抗体分子连接。充分洗涤后,在TopYield板孔中添加PCR反应液,对板壁偶联的报告DNA进行PCR扩增,间接达到检测微量禽流感病毒H5蛋白的目标。通过优化报告DNA分子和链亲和素的浓度,2种免疫PCR技术都可检测到约1fg的禽流感病毒H5蛋白,与常规ELISA方法相比,灵敏性提高了近1 000倍。

In order to detect the low concentration of avian influenza virus（AIV） in the tracheal and cloacal swabs taken from the HPAI-infected poultry,an indirect immuno-polymerase chain reaction（immuno-PCR） assay and an indirect sandwich immuno-PCR assay,based on Immunology and polymerase chain reaction（IPCR） which combining enzyme immunoassay with DNA amplification,were developed.For the indirect immuno-PCR assay,TopYield microtiter plates were coated with purified AIV/H5 protein that was recognized by anti-AIV/H5 monoclonal antibody.For the indirect sandwich immuno-PCR assay,the monoclonal antibody was immobilized on TopYield plates for detecting H5 protein that was recognized by its polyclonal antibodies.Reporter DNA was prepared by PCR amplification using biotinylated 5-primers,and it was coupled with biotinylated antibodies through streptavidin.In order to increase sensitivity and reduce background noise,the amounts of reporter DNA and streptavidin were optimized.Using the optimized concentration of reporter DNA and streptavidin,both indirect and indirect sandwich immuno-PCR assays detected AIV/H5 protein as low as 1 fg.These results are a nearly 1 000-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods.Our data demonstrate that this MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.