水稻表达HA蛋白为探针研制H9亚型禽流感抗体半定量快速检测试纸

A semi-quantitative test paper was developed for the rapid detection of H9 subtype avian influenza antibodies using rice HA protein as probe

建立一种禽流感H9亚型抗体半定量快速检测试纸,用于H9亚型禽流感(AIV)抗体的评价。含HA蛋白水稻粉末用提取液溶解后,离心,取上清,即为HA蛋白粗提液。其经过Q阴离填料和HA单抗亲和纯化,通过SDS-PAGE鉴定,重组HA蛋白纯度达到90%以上。利用胶体金标记纯化HA蛋白为探针,兔抗鸡IgG和HA的多抗分别作为检测线和质控线,依据高致病性禽流感HI诊断技术标准,建立一种适用于监测AIV H9亚型的半定量试纸检测方法。结果表明:胶体金标记HA蛋白量为30 mg/L,兔抗鸡IgG和HA多抗最佳包被质量浓度分别为1.0和1.2 g/L。试纸检测H9N2亚型阳性血清(HI为11 log_(2))的最低检出限是1∶12 800,这相当于HI效价的4 log_(2)。试纸与H5 AIV、新城疫病毒(NDV)、传染性法氏囊病病毒(IBDV)、新城疫病毒(NDV)和马立克病病毒(MDV)阳性血清以及H9亚型阴性血清无交叉反应,在室温下至少能保存6个月,与HI试验平行检测比较,两者的符合率为90.6%。因此本试验研制了一种简单、快速、准确、特异的半定量免疫层析试纸,适用于H9N2亚型抗体临床检测。

A semi-quantitative and rapid strip was developed for the evaluation of antibody against H9 subtype avian influenza(AIV).Rice powder containing HA protein was dissolved in extraction,centrifuged,and supernatant was taken,which was crude extract of HA protein.It was purified by Q Anion filler and HA monoclonal antibody,and the purity of recombinant HA protein was more than 90% by SDS-PAGE.A semi-quantitative test strip method for the detection of AIV H9 was established by using the purified HA protein as the probe,and the rabbit anti-chicken IgG and HA polyclonal antibody as the test line and quality control line,respectively,according to the diagnostic technical standards of highly pathogenic avian influenza HI.The amount of colloidal gold labeled HA protein was 30 mg/L.The optimal coating concentrations of rabbit anti-chicken IgG and HA polyclonal antibody were 1.0 and 1.2 g/L,respectively.The limit of detection for H9 N2 subtype positive sera(HI at 11 log_(2)) was 1:12 800,which corresponded to 4 log_(2) of HI titer.There was no cross-reaction with H5 ATV,Newcastle disease virus(NDV),Infectious bursal disease virus(IBDV),Newcastle disease virus(NDV),Marek’s disease virus(MDV) positive serum and H9 negative serum.It kept at room temperature for at least 6 months.Compared with HI test,the coincidence rale was 90.6%.Therefore,we develop a simple,rapid,accurate and specific seriquantitative immunochromatographic test strip in this study,which is suitable for clinical detection of H9 N2 subtype antibodies.