MOE Joint International Research Laboratory of Animal Health and Food Safety, Jiangsu Engineering Laboratory of Animal Immunology, Jiangsu Detection Center of Terrestrial Wildlife Disease, Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, People's Republic of China.
Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, 210014, People's Republic of China.
BMC Vet Res. 2021 Feb 2;17(1):64. doi: 10.1186/s12917-021-02772-6.
H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV.
The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 2, 2 and 2 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively.
In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.
H7 亚型禽流感在全球家禽业和公共卫生领域引起了极大关注。尽管血凝抑制（HI）测定法操作时间较长，但传统的血清亚型特异性诊断仍在实施。本研究基于针对 H7 禽流感病毒（AIV）血凝素（HA）蛋白的新型单克隆抗体，建立了一种用于检测针对 H7 AIV 抗体的高效、快速和高通量竞争酶联免疫吸附试验（cELISA）。
优化了抗原包被浓度、单克隆抗体浓度和血清稀释比等反应参数，用于 H7 抗体检测。采用针对 H1H9、H11H14 AIV 和其他禽病毒的抗血清对 cELISA 的特异性进行了测试。通过受试者工作特征（ROC）曲线分析，选择抑制率的截断值分别为鸡、鸭和孔雀血清的 30.11%、26.85%和 45.66%。以 HI 试验为参考方法，鸡、鸭和孔雀阳性血清的最低检测限分别达到 2、2 和 2 HI 滴度。与 HI 试验相比，该方法对 400 份临床血清样本的检测诊断准确率分别达到 100%、98.6%和 99.3%。
总之，本研究建立的 cELISA 检测方法为快速检测 H7 抗体提供了一种可靠、特异、敏感和与物种无关的血清学技术，适用于大规模血清学监测和疫苗免疫效果评估计划。
