甲型H1N1流感病毒双抗体夹心ELISA检测方法的初步建立

Development of Double-antibody Sandwich ELISA for Detecting Influenza Virus(H1N1)

建立甲型H1N1流感病毒双抗体夹心ELISA检测方法,通过优化IPTG浓度和诱导时间确定HA融合蛋白的最佳表达条件,并进行Western bloc和血凝试验鉴定。用纯化蛋白制备单克隆抗体,建立检测甲型H1N1流感病毒的双抗体夹心ELISA检测方法,对其交叉反应、符合率进行验证。结果表明,HA蛋白在BL21(DE3)中得到表达,主要以包涵体的形式存在,分子质量为64 ku,最佳诱导条件为IPTG终浓度0.2 mmol/L,诱导时间4 h。Western blot鉴定其与H1N1病毒有相同的抗原性,血凝试验表明无血凝活性。制备HA单抗并初步建立双抗体夹心ELISA检测方法,检测100份阳性PCR样本,符合率为96%。建立的夹心ELISA方法可用于H1N1亚型流感病毒的初步诊断。

To establish the Sandwich ELISA for H1N1 subtype Influenza Vilus. The best expression condition was determined by optimizing IPTG concentration and induction time. The protein was identified by Western blot and hemagglutination test. To prepare monoclonal antibody against purified recombinant protein of HA and establish the Sandwich ELISA for HIN1 subtype Influenza Vilus. Cross-reaction and coincidence rate of the method were tested. HA protein was successfully expressed with proper inducing conditions of 0.2 mmol/L IPTG and 4 hours induction in E. coli BL21（DE3）. The fusion protein was mainly in the form of inclusion body,about 64 ku, Western-blot analysis proved that the recombinant protein had good reactive ability. Monoclonal antibody against recombinant protein was prepared and the Sandwich ELISA was established preliminarily, the results from detection of 100 samples by ELISA were 96% in agreement with that of RCR test. It suggested that the Sandwich ELISA could be useful for primary diagnoses of H1N1 subtype Influenza Vilus.