双抗原夹心ELISA法检测登革病毒感染患者血清中的EDⅢ抗体

目的 建立一种检测登革病毒(dengue virus, DENV)特异性抗体的双抗原夹心ELISA方法?方法 利用毕赤酵母系统表达4个血清型登革病毒包膜蛋白Ⅲ区(envelope protein domain Ⅲ, EDⅢ),并采用改良过碘酸钠法对EDⅢ蛋白标记辣根过氧化物酶(Horseradish peroxidase, HRP),建立一种可同时检测4个血清型登革病毒EDⅢ蛋白特异性抗体的双抗原夹心ELISA法?并对2014年广州珠江医院门诊和住院确诊的登革热患者血清标本进行检测,并与澳洲Panbio MAC ELISA检测的敏感性进行比较?结果 本研究成功建立了一种检测4个血清型登革病毒EDⅢ蛋白特异性抗体的双抗原夹心ELISA法,该方法能同时检测到4个血清型登革病毒EDⅢ蛋白免疫的小鼠血清和Ⅰ型登革病毒EDⅢ蛋白免疫的兔血清,而与烟曲霉AF-MP蛋白免疫的小鼠血清和兔血清均无交叉反应?对184份健康人血清标本检测全为阴性,而对168份确诊为登革病毒感染患者血清标本检测结果表明,两种诊断方法检测结果差异无统计学意义,P<0.001(57.1% vs 57.7%),联合NS1抗原检测方法,其敏感性提高到97.6%?结论 双抗原夹心ELISA法检测登革病毒EDⅢ特异性抗体具有高度的特异性,但如果能同时联合抗原检测可明显提高登革热的早期诊断率?

Double-antigen sandwich ELISA for detecting dengue virus EDⅢ-specific antibody

This study investigated whether the EDⅢ protein of DENV1-4 is suitable for the diagnosis of dengue virus (DENV) infection. Four serotypes of DENV recombinant EDⅢ protein were expressed in Pichia pastoris and labeled with Horseradis hperoxidase(HRP) through a modified sodium periodate method. A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) for detecting EDⅢ-specific antibodies was successfully established by determining the optimal coating concentration and proportion of EDⅢ protein and HRP-EDⅢ protein for the four serotypes of DENV. The assay showed specificity to DENV1-4 EDⅢ protein immunized animal serum, with no cross-reaction with Aspergillus fumigatus MP1 immunized animal serum. The sensitivity of the double-antigen sandwich ELISA was evaluated with the Australian Panbio MAC ELISA test in sera from 168 patients with confirmed DENV infection treated at Guangzhou Zhujiang Hospital in 2014. No significant difference was found between the double-antigen sandwich ELISA and Panbio MAC ELISA (57.1% vs 57.7%, P<0􀆰001). Combining the detection results of NS1 antigen and EDⅢ antibodies markedly increased the diagnostic sensitivity from 57.1% to 97.6%. Thus, our double-antigen sandwich ELISA has high specificity in detecting EDⅢ-specific antibodies in the sera of patients with DENV infection, and simultaneous detection of antigen and antibodies can increase the diagnostic sensitivity for DENV infection.