Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus


Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

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Availability of data and material

The datasets generated and/or analyzed in the current study are available from the corresponding author on reasonable request.


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This study was supported by the National Key Research and Development Program of China (2016YFD0500800), the Forestry Science and Technology Innovation and Promotion Project of Jiangsu Province (LYKJ[2018]22), the China Agriculture Research System (CARS-40-K13), and Priority Academic Program Development of Jiangsu Higher Education Institutions.

Author information




LPY carefully conceived and designed the study. YL and JHH collected clinical serum samples, performed the experiments and analyzed data. YL and LPY wrote the manuscript. LPY, SQS, JL, ZWB and WTF revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Liping Yan.

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Conflict of interest

No potential financial or non-financial conflict of interest was declared by the authors.

Ethical approval

The animals in this study were treated in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. All sample collection procedures in the current study were scrutinized and authorized by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (approval. no. SYXK (Su) 2017-0007).

Consent to participate

Written informed consent to use 104 clinical serum samples, which were collected from breeding poultry farms in Jiangsu province of China, was obtained from the owner of the animals. All efforts were made to minimize animal suffering during sample collection.

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Supplementary Information

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Supplementary Fig. S1 SDS-PAGE analysis of the expression and purification of recombinant pET-32a-AIV-NP (a), pCold-І-NDV-P (b), and pGEX-4T-1-IBV-nsp5 (c) after transformation of E. coli BL21. M, standard protein marker; lane 1, uninduced E. coli transformed with pET-32a, pCold І, or pGEX-4T-1; lane 2, induced E. coli transformed with pET-32a, pCold І, or pGEX-4T-1; lane 3, uninduced E. coli transformed with pET-32a-AIV-NP, pCold-І-NDV-P, or pGEX-4T-1-IBV-nsp5; lane 4, induced E. coli transformed with pET-32a-AIV-NP, pCold-І-NDV-P, or pGEX-4T-1-IBV-nsp5; lane 5, purified AIV-NP, NDV-P, or IBV-nsp5 protein. Fig. S2 Western blot verification of the immunoreactivity of an AIV-positive serum with AIV NP (a), NDV-positive serum with NDV-P (b), and IBV-positive serum with IBV nsp5 (c). M, standard protein marker; lane 1, purified AIV NP, NDV P or IBV nsp5 fusion protein; lane 2, induced E. coli transformed with pET-32a, pCold І, or pGEX-4T-1. Fig. S3 The limit of detection for AIV NP, NDV P, and IBV nsp5 protein determined by testing 50 standard negative chicken sera. Each dot represents one negative chicken serum sample. Fig. S4 Stability of the triplex protein microarray slides. The signal intensities of AIV NP, NDV P and IBV nsp5, positive control, and negative control spots were compared between freshly prepared slides and slides stored at 4 °C for six months (PDF 295 KB)

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Li, Y., Hu, J., Lei, J. et al. Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus. Arch Virol 166, 1113–1124 (2021).

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