ŁUKASIEWICZ Research Network - Industrial Chemistry Institute, Rydygiera 8 Street, 01-793, Warsaw, Poland.
Virol J. 2021 Apr 30;18(1):91. doi: 10.1186/s12985-021-01564-6.
H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA).
In the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1-H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis.
The H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively.
The H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.
H5 亚型高致病性（HP）禽流感病毒（AIV）在禽类中可导致高死亡率，并在人类中偶发感染，且常导致死亡，而 H5N1 病毒具有大流行的潜力。由于具有兽医和公共卫生意义，这些高致病性 AIV 以及倾向于突变形成 HP 病毒的低致病性（LP）H5 亚型 AIV 都受到流行病学监测，并必须向世界动物卫生组织（OIE）报告。我们之前的工作提供了一组独特的针对 H5 血凝素（HA）的 6 种不同单克隆抗体（mAb），这满足了监测家禽中 AIV 感染和疫苗接种的高特异性工具的需求。在这项研究中，我们选择了其中一种 mAb 来开发用于检测鸡血清中 H5 亚型特异性抗体的表位阻断（EB）ELISA（H5 EB-ELISA）。
在 H5 EB-ELISA 中，使用杆状病毒表达载体系统产生的 H5 HA 蛋白作为包被抗原，G-7-27-18 mAb 作为阻断抗体。通过检测 358 份来自未免疫鸡和用 AIV 的 H1-H16 亚型或重组 H5 HA 抗原免疫的鸡的血清，分别获得参考和实验性抗血清，评估该检测方法的性能特征。根据血凝抑制（HI）检测的结果（亚型特异性血清诊断的金标准），将样本分类为抗 H5 HA 阳性或阴性。
H5 EB-ELISA 正确地区分了抗 H5 HA 阴性血清，包括针对非 H5 亚型 AIV 的血清，以及针对各种来源的 H5 HA 的抗体阳性血清。初步验证显示，该检测方法的分析特异性为 100%，诊断特异性为 97.6%，用于检测参考和实验性抗血清中的抗 H5 HA 抗体时，诊断敏感性分别为 98.0%和 99.1%。
H5 EB-ELISA 在诊断评估方面表现良好。因此，需要使用更大的鸡血清集和接受者操作特征（ROC）分析来进一步优化和验证该检测方法。此外，本研究提供了开发用于鸟类的多物种筛选检测或用于人类的诊断检测的有价值的基础。
