Advertisement

A new nanobody-enzyme fusion protein-linked immunoassay for detecting antibodies against influenza A virus in different species

  • Author Footnotes
    ‡ The two authors contributed equally to the paper.
    Pinpin Ji
    Footnotes
    ‡ The two authors contributed equally to the paper.
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Author Footnotes
    ‡ The two authors contributed equally to the paper.
    Kun Wang
    Footnotes
    ‡ The two authors contributed equally to the paper.
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Lu Zhang
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Zhenda Yan
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Min Kong
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Xuwen Sun
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Qiang Zhang
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Ning Zhou
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Baoyuan Liu
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • En-Min Zhou
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Yani Sun
    Correspondence
    Correspondences:
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Xinjie Wang
    Correspondence
    Correspondences:
    Affiliations
    Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518100, China
    Search for articles by this author
  • Qin Zhao
    Correspondence
    Correspondences:
    Affiliations
    Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China
    Search for articles by this author
  • Author Footnotes
    ‡ The two authors contributed equally to the paper.
Open AccessPublished:November 16, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102709

      Abstract

      Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAVs infection in different species. Recently, nanobodies, which show advantages of easy gene-editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nanobodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can cross-react with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen, as well as dilutions of the fusion and testing sera were 100 ng/well, 1:4,000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. Additionally, we found that the cELISA and hemagglutination inhibition (HI) test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species.

      Keywords

      Introduction

      Influenza is an acute infectious disease caused by the influenza virus (IV), which can rapidly evolve and can cause significant morbidity and mortality (
      • Cox N.J.,S.K.
      ,
      • Wille M.
      • Holmes E.C.
      The Ecology and Evolution of Influenza Viruses.
      ). The IV, belonging to the Orthomyxoviridae family, is a negative-sense, single-stranded segmented RNA, and an enveloped virus. The complete genome encodes RNA polymerase subunits, nucleoprotein (NP), matrix protein (M1), membrane protein (M2), nonstructural protein (NS1), nuclear export protein (NEP), hemagglutinin (HA), and neuraminidase (NA) (
      • Webster R.G.
      • Govorkova E.A.
      Continuing challenges in influenza.
      ,

      Palese, P., and Young, J. F. (1982) Variation of Influenza A, B, and C Viruses. 215, 1468-1474

      ,
      • Saczynska V.
      • Florys-Jankowska K.
      • Porebska A.
      • Cecuda-Adamczewska V.
      A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin.
      ). Based on the identity of the internal conserved proteins including NP and M1 proteins, the IVs are divided into type A, B, C, and D (
      • Webster R.G.
      • Bean W.J.
      • Gorman O.T.
      • Chambers T.M.
      • Kawaoka Y.
      Evolution and ecology of influenza A viruses.
      ) of which, influenza A viruses (IAVs) are further divided into 18 H subtypes (H1-H18) and 11 N subtypes (N1-N11), according to their HA and NA makeup (

      Animals, W. O. f. A. H. M. o. D. T. a. V. f. T. (2018) Chapter 3.3.4. Avian influenza (infection with avian influenza viruses).

      ,
      • Nobusawa E.
      • Aoyama T.
      • Kato H.
      • Suzuki Y.
      • Tateno Y.
      • Nakajima K.
      Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses.
      ). The highly variable IAVs are spread across a wide range of mammalian and avian species, such as humans, pigs, horses, dogs, cats, minks, seals, whales, and domestic and wild birds (

      Palese, P., and Young, J. F. (1982) Variation of Influenza A, B, and C Viruses. 215, 1468-1474

      ,

      Sreenivasan, C. C., Thomas, M., Kaushik, R. S., Wang, D., and Li, F. (2019) Influenza A in Bovine Species: A Narrative Literature Review. Viruses 11

      ). All subtypes of IAVs infect aquatic birds, which provide the genetic diversity required for the spread of IAVs across species (
      • Webster R.G.
      • Bean W.J.
      • Gorman O.T.
      • Chambers T.M.
      • Kawaoka Y.
      Evolution and ecology of influenza A viruses.
      ) (
      • Matrosovich M.N.
      • Matrosovich T.Y.
      • Gray T.
      • Roberts N.A.
      • Klenk H.-D.
      Human and avian influenza viruses target different cell types in cultures of human airway epithelium.
      ). Birds and pigs play important roles in global influenza pandemics caused by genomic reassortment and are the key hosts for the spread of influenza across the interspecies barrier (
      • Wille M.
      • Holmes E.C.
      The Ecology and Evolution of Influenza Viruses.
      ,
      • Hay A.J.
      • Gregory V.
      • Douglas A.R.
      • Lin Y.P.
      The evolution of human influenza viruses.
      ). Therefore, monitoring the history of IAVs immunity and the infection of different susceptible species, especially in birds and pigs, is urgently needed for global IAVs surveillance and control (
      • Abolnik C.
      • Fehrsen J.
      • Olivier A.
      • van Wyngaardt W.
      • Fosgate G.
      • Ellis C.
      Serological investigation of highly pathogenic avian influenza H5N2 in ostriches (Struthio camelus).
      ,
      • Li Y.
      • Hu J.
      • Lei J.
      • Fan W.
      • Bi Z.
      • Song S.
      • Yan L.
      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.
      ).
      Many serologic testing assays have been developed for detecting anti-IAV antibodies in the serum samples. These assays include hemagglutination inhibition (HI), agar gel immunodiffusion (AGID), virus neutralization test (VNT), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA) (
      • Saczynska V.
      • Florys-Jankowska K.
      • Porebska A.
      • Cecuda-Adamczewska V.
      A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin.
      ,
      • Li Y.
      • Ye H.
      • Liu M.
      • Song S.
      • Chen J.
      • Cheng W.
      • Yan L.
      Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus.
      ). Of these, ELISA has advantages with high sensitivity, specificity, accuracy, and a high throughput, and is thus widely used for early diagnosis and screening (
      • Abolnik C.
      • Fehrsen J.
      • Olivier A.
      • van Wyngaardt W.
      • Fosgate G.
      • Ellis C.
      Serological investigation of highly pathogenic avian influenza H5N2 in ostriches (Struthio camelus).
      ,
      • Honda T.
      • Gomi S.
      • Yamane D.
      • Yasui F.
      • Yamamoto T.
      • Munakata T.
      • Itoh Y.
      • Ogasawara K.
      • Sanada T.
      • Yamaji K.
      • Yasutomi Y.
      • Tsukiyama-Kohara K.
      • Kohara M.
      Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies.
      ). Since surface glycoproteins (HA and NA) of IAVs have a high rate of antigenic variability, it is problematic to develop a universal method for detecting antibodies against different subtypes IAV using them as antigens (
      • Yang M.
      • Berhane Y.
      • Salo T.
      • Li M.
      • Hole K.
      • Clavijo A.
      Development and application of monoclonal antibodies against avian influenza virus nucleoprotein.
      ). However, as we known, the NP is conserved among different subtypes IAV. In addition, although the percentage of anti-NP antibodies is generally lower than the one of anti-HA antibodies, the antibodies against NP can also be quickly detected after IAV infected hosts. So, the NP was also widely used and one of the most promising diagnostic targets for IAVs infection (

      Shu, L. L., Bean, W. J., and Webster, R. G. J. J. o. V. (1993) Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. 67, 2723-2729

      ,
      • Tu Y.-C.
      • Chen K.-Y.
      • Chen C.-K.
      • Cheng M.-C.
      • Lee S.-H.
      • Cheng I.-C.
      Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying.
      ).
      There are indirect and competitive ELISAs (cELISAs), which use the highly conserved IAV-NP protein as an antigen to detect antibodies against different subtypes of IAVs in different species (
      • Hu Y.
      • Sneyd H.
      • Dekant R.
      • Wang J.
      Influenza A Virus Nucleoprotein: A Highly Conserved Multi-Functional Viral Protein as a Hot Antiviral Drug Target.
      ). However, most commercially available ELISA kits were produced based on the enzyme-conjugated second or monoclonal antibody. As the production of enzyme-labeled antibodies is complicated and shows a considerable degree of variation between lots, these kits are high in cost and make it difficult to control the consistency from batch to batch (
      • Du T.
      • Zhu G.
      • Wu X.
      • Fang J.
      • Zhou E.-M.
      Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.
      ) (
      • Goodell C.K.
      • Prickett J.
      • Kittawornrat A.
      • Johnson J.
      • Zhang J.
      • Wang C.
      • Zimmerman J.J.
      Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine.
      ).
      Nanobody, or named single-domain antibodies, are a novel type of engineered antibodies derived from the variable domain of the heavy chain of heavy-chain antibody (VHH) (
      • Muyldermans S.
      • Lauwereys M.
      Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies.
      ). Nanobodies have certain advantages, including their small molecular weight (15 kDa) and their suitability for genetic manipulation. Thus, they are widely used in disease diagnosis, surveillance, and prevention (
      • Muyldermans S.
      • Lauwereys M.
      Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies.
      ) (
      • Li D.
      • Ji F.
      • Huang C.
      • Jia L.
      High Expression Achievement of Active and Robust Anti-β2 microglobulin Nanobodies via E.coli Hosts Selection.
      ). Regarding the development of disease diagnosis kits, derivatives can be easily established by coupling the reporters, and this is fiscally favorable compared to conventional antibodies (
      • Wrapp D.
      • De Vlieger D.
      • Corbett K.S.
      • Torres G.M.
      • Wang N.
      • Van Breedam W.
      • Roose K.
      • van Schie L.
      • Team V.-C.C.-R.
      • Hoffmann M.
      • Pohlmann S.
      • Graham B.S.
      • Callewaert N.
      • Schepens B.
      • Saelens X.
      • McLellan J.S.
      Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies.
      ). For example, the reporter-nanobody fusion protein (RANbody) has been applied in the development of detection assays, and for labeling cells and tissues (
      • Ren W.
      • Li Z.
      • Xu Y.
      • Wan D.
      • Barnych B.
      • Li Y.
      • Tu Z.
      • He Q.
      • Fu J.
      • Hammock B.D.
      One-Step Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Fusion for Detection of Aflatoxin B(1) in Cereal.
      ) (
      • Lu Q.
      • Li X.
      • Zhao J.
      • Zhu J.
      • Luo Y.
      • Duan H.
      • Ji P.
      • Wang K.
      • Liu B.
      • Wang X.
      • Fan W.
      • Sun Y.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays.
      ). Recently, the nanobody-horseradish peroxidase (HRP) fusion was universally used to develop immunoassays to detect antibodies in the sera (
      • Du T.
      • Zhu G.
      • Wu X.
      • Fang J.
      • Zhou E.-M.
      Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.
      ,
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ). However, few studies have developed an immunoassay for detecting anti-IAV antibodies in the sera from different species using nanobodies as reagents.
      In the present study, the NP from a subtype of the H9N2 avian influenza virus (H9N2 AIV, H9N2-NP) was expressed using the Escherichia coli (E. coli) system. Then, H9N2-NP was used as an antigen to immunize the Bactrian camel and to screen the nanobodies obtained. In total 5 nanobodies against H9N2-NP were obtained and were modified to contain an HRP tag. Subsequently, the cross-reactivity of these nanobodies with other subtypes of IAVs was assessed, and a nanobody recognizing a common epitope located in the NP of all IAV subtype strains was characterized. Then, using this nanobody-HRP fusion as a reagent, a cELISA was developed to qualitatively detect anti-IAV antibodies in the sera from different species. Our study provided a type of cELISA with rapid and easy operation for the diagnosis of IAV infection in different species. Importantly, the method produced was low in cost for following commercial production without the HRP-labeled antibodies in vitro.

      Results

      Expression and purification of the recombinant H9N2-NP protein

      To immunize the camels, anti-H9N2-NP nanobodies were screened and the cELISA was established; the recombinant H9N2-NP protein was expressed using a bacterial system. SDS-PAGE analysis showed that the recombinant protein was successfully expressed and purified using a Ni-NTA column with the expected size of 56 kDa (Figure S1A). Western blotting analysis revealed that the recombinant H9N2-NP protein specifically reacted with positive chicken serum samples for anti-H9N2 AIV antibodies (Figure S1B), indicating the recombinant proteins possessed antigenicity.

      Construction of a phage display VHH library against the H9N2-NP protein

      After the camel was immunized with the H9N2-NP protein, the titer of antibody against the protein in serum samples reached 1:128,000 based on iELISA (Figure 1A). Next, a phage display library containing 9.8 × 108 individual transformants was successfully constructed using the PBLs from the immunized camel. Subsequently, the results showed that the positive rate of the library was 98% by colony PCR assay (Figure 1B) and each clone from the selected 96 clones contained a different VHH sequence (data not shown). The high capacity and positivity rate of the library confirmed the suitable heterogeneity of the library.
      Figure thumbnail gr1
      Figure 1Screening of the nanobodies against the recombinant H9N2-NP protein. A, Titers of antibodies against H9N2-NP protein in the sera from the camel after the fifth immunization. B, Identification of the positive rate for the VHH library by PCR. C, Detection of the periplasmic extracts from 96 clones reacting with H9N2-NP by indirect ELISA. D, Alignment of the amino acid sequences of the 5 screened nanobodies. Numbering and CDRs are in accordance with previous methods. E, Determination of specific reactions between the 5 screened nanobodies and the H9N2-NP protein.

      Screening of nanobodies against H9N2-NP protein.

      After three rounds of panning, the results showed that the phage particles carrying H9N2-NP-specific VHH were significantly enriched (Table S2). Using the iELISA to detect the nanobodies in the periplasm from the 96 clones, the results showed that 94 clones could react with the H9N2-NP protein (Figure 1C). These 94 positive clones were sequenced and five nanobodies were obtained based on the CDR3 region of the sequences. They were named H9N2-NP-Nb5, -Nb16, -Nb18, -Nb42, and -Nb53. Alignment results suggested that the five nanobodies possessed typical hydrophilic amino acid substitutions in the frame-work-2 regions (Figure 1D). In addition, the five nanobodies were tested by iELISA using H9N2-NP and NDV-NP separately as coating antigens. The results showed that they specifically reacted with the H9N2-NP (Figure 1E), but not with NDV-VP which was expressed with the same procedures and systems as H9N2-NP (
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ).

      Expression of the five nanobody-HRP fusion proteins against H9N2-NP protein

      Based on the above descriptions, the five anti-H9N2 nanobodies fusing with HRP were successfully expressed using HEK-293T cells as confirmed by IFA identification (Figure 2A). Using the supernatant from the transfected HEK-293T cells as testing antibodies, the results of direct ELISA showed that all five nanobody-HRP fusion proteins were secreted and were subsequently termed H9N2-NP-Nb5-HRP, -Nb16-HRP, -Nb18-HRP, -Nb42-HRP, and -Nb53-HRP (Figure 2B). The titer of the H9N2-NP-Nb5-HRP fusion proteins in the supernatants was 1:10,000, and the titers of the other four H9N2-NP-nanobodies-HRP fusions were 1:1,000 (Figure 2C).
      Figure thumbnail gr2
      Figure 2Identification of five anti-H9N2-NP nanobodies with HRP fusion protein secretion expressed in HEK-293T cells. A, Detection of the H9N2-NP-Nb5, -Nb16, -Nb18, -Nb42, -Nb53-HRP fusions expressed in HEK-293T cells by IFA. A representative image of H9N2-Nb5-HRP is shown, and similar results were observed for the other 4 nanobodies-HRP against H9N2-NP. B, Analysis of the binding of H9N2-NP-Nb5, -Nb16, -Nb18, -Nb42, and -Nb53-HRP fusions with H9N2-NP by direct ELISA. C, Titers of the H9N2-NP-Nb5, -Nb16, -Nb18, -Nb42, -Nb53-HRP fusions in the medium of HEK293T cells using direct ELISA.

      Cross-reaction between nanobody-HRP fusion proteins and other IAVs subtypes

      To analyze the five nanobody-HRP fusion proteins cross-reaction with other subtypes IAV and IBV, the direct ELISAs using the NP of H1N1, H3N2, B/Yamagata/16/1988 and killed denatured H5N1 and H7N9 particles as the coating antigens was performed. SDS-PAGE analysis showed that the NP of H1N1, H3N2 and B/Yamagata/16/1988 were successfully expressed using the E. coli system with the expected size and was successfully purified using a Ni-NTA column (Figure 3A). The results of direct ELISA showed that the H9N2-Nb5, -Nb16, -Nb18, -Nb42, and -Nb53-HRP could cross-react with the NP proteins of subtypes H1N1, H3N2, H5N1, and H7N9 IAVs, but not react with the NP of B/Yamagata/16/1988 (Figure 3B). In addition, each positive serum sample for anti-H9N2, H1N1, H3N2, H5N1, and H7N9 antibodies confirmed using the commercial IDEXX ELISA kit was detected using these nanobody-HRP fusions as reagents by blocking ELISA. The results showed that the PI of cELISA using H9N2-NP-Nb5-HRP as a probe reached 80% (Figure 3C). The above results indicated that the epitope recognized by the H9N2-NP-Nb5 may be conserved across different subtypes of IAVs. Based on these results, H9N2-NP-Nb5-HRP was selected for identification of its recognizing epitope to analyze the conservation in all subtypes of IAVs and for developing the cELISA.
      Figure thumbnail gr3
      Figure 3Analysis of cross-reactivity between the five nanobody-HRP fusion proteins and other subtypes of IAVs. A, SDS-PAGE analysis of H1N1-NP, H3N2-NP and IBV-NP expressed using the E. coli system. M, protein molecular markers; lane 1, H1N1-NP; lane 2, H3N2-NP, lane 3, IBV-NP. B, Analysis of H9N2-NP-Nb5, -Nb16, -Nb18, -Nb42, -Nb53-HRP cross-reacting with H1N1-NP, H3N2-NP, IBV-NP and killed viral particles of H5N1 and H7N9 IAV by direct ELISA. C, Analysis of the binding of five nanobody-HRP fusions to H9N2-NP blocked by the positive sera being separately for antibodies against H9N2 (S1), H1N1 (S2), H3N1 (S3), H5N1 (S4) and H7N9 (S5) using blocking ELISA.

      Recognition of the conserved epitope recognized by H9N2-NP-Nb5-HRP among the different subtypes of IAV strains

      To identify the epitope recognized by H9N2-NP-Nb5-HRP, different truncated and overlapping H9N2-NP fragments were designed (Figure 4A). After these fragments were expressed using the E. coli system, SDS-PAGE and western blotting analysis showed that they were successfully expressed with the expected size and purified by a Ni-NTA column (Figure 4B and C). The results of direct ELISA using these truncated fragments as coating antigens showed that the H9N2-NP-Nb5-HRP reacted with fragments spanning 1-1,308 bp (N436 aa), 1-1,215 bp (N405 aa), 1-1,182 bp (N394 aa) and 1-1,167 bp (N389 aa), but not with 1-747 bp (N249 aa), 747-1,494 bp (C249 aa), 1-1,122 (N374 aa), 373-1,494 (C374 aa), 187-1,494 bp (C436 aa), or 1-1,152 bp (N384 aa) (Figure 4D). These results suggested that the epitope recognized by H9N2-NP-Nb5-HRP may be a native conformational epitope and the two regions of aa 1-62 and aa 385-389 may be important domains for maintaining the conformation.
      Figure thumbnail gr4
      Figure 4Precise definition of the minimal motif recognized by H9N2-NP-Nb5-HRP. A, Schematic diagram of different H9N2-NP truncated fragments. B, SDS-PAGE analysis of different fragments expressed using the E. coli system. M, protein molecular markers; lane 1 to 11: full length of H9N2-NP, 1-747 bp (N249 aa), 747-1,494 bp (C249 aa), 1-1,122 (N374 aa), 373-1,494 (C374 aa), 1-1,308 bp (N436 aa), 187-1,494 bp (C436 aa), 1-1,215 bp (N405 aa), 1-1,152 bp (N384 aa), 1-1,167 bp (N389 aa), and 1-1,182 bp (N394 aa). C, Western blotting analysis of expression of different fragments using anti-His monoconal antibodies as the primary antibody. Lane 1 to 11: Same as panel b. D, Analysis of H9N2-NP-Nb5-HRP reacting with H9N2-NP and different truncated fragments of H9N2-NP by direct ELISA.
      To precisely define the epitope, AlphaFold2 server was used to predict the structure of proteins. The docking model showed that the W95, T96, and K100 are located in the H9N2-NP-Nb5 regions aa 354-356, aa 385-387, and aa 445-449 in the H9N2-NP were essential for H9N2-NP-Nb5-HRP interaction with the H9N2-NP protein (Figure 5A). Based on the reaction of different fragments and predicted results, the amino acids located in aa 388-389 (H9N2-NP388-389M), aa 385-389 (H9N2-NP385-389M), and the three regions (H9N2-NP3M) predicted by the docking model were mutated into alanine (A) and expressed using the E. coli system. In addition, the W95, T96, and K100 of H9N2-NP-Nb5-HRP were also all mutated to A (termed H9N2-NP-Nb53M-HRP). SDS-PAGE and western blotting showed that the mutants of H9N2-NP were successfully expressed with the expected sizes (Figure 5B and C). Furthermore, the direct ELISA results showed that H9N2-NP3M did not react with H9N2-NP-Nb5-HRP, and H9N2-NP-Nb53M-HRP did not react with H9N2-NP. Additionally, although H9N2-NP385-389M still reacted with H9N2-NP-Nb5-HRP, the OD450nm value of direct ELISA using the coated antigen was significantly lower than that of H9N2-NP (Figure 5D). So, the results of direct ELISA using the mutated proteins indicated that the predicted amino acids for interaction between H9N2-NP and H9N2-NP-Nb5-HRP were correct. Furthermore, these results also suggested that the epitope recognized by the H9N2-NP-Nb5-HRP was a native conformational epitope.
      Figure thumbnail gr5
      Figure 5Identification of conserved epitopes recognized by H9N2-NP-Nb5-HRP among the different subtypes of IAV strains. A, Structure of the predicted docking complex between H9N2-NP-Nb5 and H9N2-NP protein. The H9N2-NP protein is shown in blue and H9N2-NP-Nb5 is shown in brown. B, SDS-PAGE analysis of different mutated H9N2-NP proteins expressed using the E. coli system. M, protein molecular markers; lanes 1 to 4, H9N2-NP, H9N2-NP388-389M, H9N2-NP385-389M, and H9N2-NP3M. C, Western blotting to identify the expression of different mutated proteins with anti-His monoclonal antibodies. Lanes 1 to 11: Same as the panel b. D, Determination of different mutated or wild-type H9N2-NP reaction with wild-type or mutated H9N2-NP-Nb5-HRP by direct ELISA. E, Sequence alignments of the key motifs binding to H9N2-NP-Nb5 among different IAV strains.
      To further analyze the amino acid conservation of the epitope, the aa 354-356, aa 385-387, and aa 445-449 of the NP from different IAV strains were aligned. Sequence alignments showed that the epitope was highly conserved among the different subtypes of IAV strains (Figure 5E).

      Development of the cELISA using H9N2-NP-Nb5-HRP fusion protein as a probe

      The conditions of cELISA using the H9N2-NP-Nb5-HRP fusion as the probe was optimized. Using checkerboard titration experiments with the direct ELISA, the results showed that the optimized amount of coated antigen was 100 ng/well and the optimal dilution of H9N2-NP-Nb5-HRP was 1:4,000 (Table S3). Using the different dilutions of positive and negative sera for detection with the cELISA, the results showed that the optimized dilution of testing sera was 1:10 (Table S4). Finally, the results for determining the optimal times showed that the optimized incubation time between the mixture of H9N2-NP-Nb5-HRP with testing sera and coated antigen was 20 min and the optimal color reaction time was 10 min (Table S5).

      Cut-off value of the cELISA

      The average PI (X) value of 180 negative sera from humans, pigs, and chickens detected by the developed cELISA was 1.0%, and the SD was 4.4%. The cut-off value of the cELISA was 14.2% (1.0% + 3SD), indicating that the PI values of the tested sera by the cELISA were more than 14.2%, suggesting that the serum sample was positive for the anti-IAV antibody. If the PI value was less than 14.2%, this would then suggest they were negative.

      Sensitivity, specificity, reproducibility, and stability of the cELISA

      The results of testing different dilutions of positive sera for anti-IAV antibodies with the cELISA showed that the sera at a dilution of 1:640 was negative, and those at 1:320 were all positive (Figure 6A). These indicated that the limits of detection of the cELISA for testing sera was 1:320.
      Figure thumbnail gr6
      Figure 6Sensitivity, specificity, and stability of the nanobody-based cELISA for detecting anti-AIV antibodies. A, Determination of the largest dilution of positive sera for anti-AIV antibodies. B, Cross-reaction of cELISA by detecting antibodies against anti-chicken, pig, and human other viruses. C, Analysis of the stability of the cELISA.
      The results of testing the positive sera for anti-other chicken, pig, and human viruses’ antibodies showed that the PI values of these sera were all below 14.2%, suggesting that the specificity of cELISA was good (Figure 6B).
      To analyze the reproducibility of the cELISA, five positive and negative sera were tested to evaluate the intra-assay and inter-assay variabilities. The intra-assay CV of the PI was analyzed in the range of 3.14%-7.02% with a median value of 5.08%, while the range for the inter-assay CV was 4.69%-12.73% with a median value of 8.71% (Table S6). These data indicate that the cELISA method exhibited good reproducibility.
      The results of direct ELISA using the plates and H9N2-NP-Nb5-HRP stored at 4°C for 0, 30, 60, 90, 120, 150, and 180 days showed that the OD450nm values were ∼1.0, indicating that the plates and the competitive agents H9N2-NP-Nb5-HRP remained active. The results of cELISA showed that the PI of cELISA stabilized at 80%, suggesting that the assay exhibited good stability over the past 180 days (Figure 6C).

      Application of the cELISA to detect anti-IAV antibodies in the different species

      To evaluate the assay for detecting anti-IAV antibodies in different species, the 2,155 sera samples from human (n = 660), swine (n = 660), pet dog (n = 524), goat (n = 195), cattle (n = 28), rabbit (n = 44), cat (n = 44), pet dog (n = 524), duck (n = 98), and wild birds (n = 63) were simultaneously tested by the developed cELISA and the commercial IDEXX ELISA kit. For human sera, the positive rates of the cELISA and the commercial IDEXX ELISA kit were 97.58% (644/660) and 97.88% (646/660), respectively. For pig serum samples, the positive rates of the cELISA and the commercial IDEXX ELISA kit were 82.42% (544/660) and 83.33% (550/660), respectively. For pet dogs, the positive rates were 25.19% (132/524) and 25.95% (136/524), respectively. The positive rates of goat sera were 14.36% (28/195) and 14.87% (19/195), respectively. For cattle, the positive rates for both kits were 46.43% (13/28), and the positive rates of rabbit sera for both kits were 31.82% (14/44). For cat sera, the positive rates were 2.27% (1/44) and 6.82% (3/44), respectively. The positive rates of duck sera were 87.31% (227/260) and 88.08% (229/260), respectively, and for the wild birds, the rates were 27.73% (33/119) and 31.93% (38/119), respectively (Table 1). The above results indicated that the developed cELISA could be used to detect anti-IAV antibodies in different species.
      Table 1Detection of anti-IAV antibodies in the different species using the developed cELISA.
      SpeciescELISANumbers of seraCommercial IDEXX ELISA kitAgreementKappa ValuePositive rate
      +-
      Human+644644099.7%0.9397.6%
      -16214
      Pig+544544099.1%0.9782.4%
      -1166110
      Pet dog+132131198.9%0.9725.2%
      -3925387
      Goat+2826297.4%0.9014.4%
      -1673164
      Cattle+13130100%1.046.4%
      -15015
      Rabbit+14140100%1.031.8%
      -30030
      Cat+11095.5%0.482.3%
      -43241
      Duck+227224396.9%0.8687.3%
      -33528
      Wild bird+3330390.8%0.7827.7%
      -86878

      Comparisons of the analytical performance among the cELISA, commercial ELISA kit, and HI test

      A total of 185 clinical chicken sera and 91 sequential sera from the SPF chickens challenged with H9N2 AIV were tested by the cELISA, HI test, and commercial IDEXX ELISA kit. For the clinical chicken sera, the positive rates of the three assays were 57.30%, 57.30%, and 51.35%, respectively (Table 2). When the performance of three methods was compared, the cELISA allowed to identify 106 positive and 79 negative serum samples, while in the commercial IDEXX ELISA only 90 were positive and 16 showed to be negative. When the 79 cELISA-negative samples were subjected to the commercial ELISA, 5 of them were positive and 74 were negative. The agreements were 90.4% between the cELISA and the commercial IDEXX ELISA kit (Table 2). The results of both cELISA and HI test coincided in 106 of the 185 serum samples with an agreement rate of 100%. In addition, the statistical analysis showed that there were no significant differences (κ values were >0.4) between the cELISA and either the HI test (κ values = 1.0) or the commercial IEDXX ELISA kit (κ values = 0.772). For the challenged chicken sera, the results showed that the positive rates were 73.63% (67/91), 61.54% (56/91), and 59.34% (54/91), respectively (Table 2). The agreements were 85.71% between the cELISA and the commercial IDEXX ELISA kit, and 87.91% between the cELISA and HI test (Table 2).
      Table 2Comparison of the cELISA with HI test developed in the present study with the commercial ELISA using clinical samples and challenged chicken serum samples
      SamplescELISANumbersCommercial IDEXX ELISA kitAgreement (%)Kappa ValueHI testAgreement (%)Kappa Value
      +-+-
      Clinical chickens+106901690.40.7710601001.0
      -79574079
      Challenged chickens+67541385.70.69561187.90.73
      -24024024
      Additionally, the sensitivities of three approaches were also compared. All sera from 7 days post infection (dpi) were positive using cELISA, but only 4 samples were positive with the HI test and 2 samples were positive with the commercial IEDXX ELISA kit (Figure 7A). At 5 dpi, one sample was positive using the cELISA, while all sera were negative with the HI test and commercial IDEXX ELISA kit (Figure 7B). These results suggested that the developed cELISA was higher in sensitivity than the HI test and commercial IDEXX ELISA kit for detecting anti-IAV antibodies from challenged chicken sera.
      Figure thumbnail gr7
      Figure 7Comparisons of the developed cELISA using an HI test and using a commercial ELISA kit by detecting sequential sera from the SPF chickens challenged with H9N2 AIV stock. A, Detection of antibodies against H9N2 AIV in the serial sera from the challenged SPF chickens with the cELISA and a commercial IDEXX ELISA kit. B, Detection of the above serial sera with the cELISA and HI assays.

      Discussion

      Antibodies have proven to be central to the development of diagnostic methods, moving from polyclonal antibodies to the milestone development of monoclonal antibodies (mAbs). With the rapid development of mAb technology, many modern immunoassays have been developed and applied in a wide range of biological fields (
      • Gao Y.
      • Huang X.
      • Zhu Y.
      • Lv Z.
      A brief review of monoclonal antibody technology and its representative applications in immunoassays.
      ). Of these, ELISA has several advantages including high sensitivity, strong specificity, convenience, and fewer specific requirements with regard to experimental conditions and personnel, making it suitable for large-scale rapid screening of influenza immunity, and epidemic history (
      • Lequin R.M.
      Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA).
      ). Competitive and blocking ELISAs to detect antibodies are useful methods for zoonotic diseases (or those that infect a wide range of hosts), as different secondary antibodies are required for the detection of antibodies from different species using indirect ELISA. This is particularly true for the sera from certain rare species, for which the preparation of secondary antibodies is challenging (
      • Gao Y.
      • Huang X.
      • Zhu Y.
      • Lv Z.
      A brief review of monoclonal antibody technology and its representative applications in immunoassays.
      ). At present, most ELISA kits are developed using mAbs as detection reagents. Based on traditional antibodies, although the competitive and blocking ELISAs avoid the use of secondary antibodies against different species, they require in vitro labeling of mAbs, resulting in poor batch-to-batch consistency (

      Duan, H., Chen, X., Zhao, J., Zhu, J., Zhang, G., Fan, M., Zhang, B., Wang, X., Sun, Y., Liu, B., Zhou, E.-M., Zhao, Q., and Theel Elitza, S. Development of a Nanobody-Based Competitive Enzyme-Linked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses. Journal of clinical microbiology 59, e01580-01521

      ). In addition, the large size of mAbs makes them a challenging substrate for recombinant gene editing, and the high cost of production technology limits their development to some extent (
      • Kumar V.
      • Mishra B.
      Monoclonal antibodies and its applications: An overview.
      ). Nanobodies are relatively novel diagnostic probes with lower molecular weights than traditional mAbs, with in vitro recombination and protein expression methods using bacterial production systems (
      • Pillay T.S.
      • Muyldermans S.
      Application of Single-Domain Antibodies ("Nanobodies") to Laboratory Diagnosis.
      ). Additionally, the simpler gene structure of nanobodies allows for easier modification using fusion constructs with reporter genes, which greatly simplifies the commercial production of ELISA diagnostic kits. In this study, based on these advantages, five anti-H9N2 nanobodies were screened and a nanobody-HRP fusion protein recognizing the common epitope of all subtypes of IAV strains was selected to establish a cELISA for detecting anti-IAV antibodies in the different species. The developed cELISA based on the nanobody fusion HRP protein not only overcame the above short comings, but also showed good sensitivity, specificity, and agreement with a commercial ELISA kit. The developed assay utilized the advantages of nanobody fusion with HRP, which simplified the operational steps and shortened the detection time (∼40 min in total). More importantly, the diagnostic kit could be assembled by directly collecting the culture supernatant of the cell line stably expressing the nanobody-HRP fusion protein without antibody labeling, alleviating the need for the complex preparation process of species-specific secondary antibodies and inconsistency between batches, whilst also reducing the cost of commercial production. The developed cELISA could detect anti-IAV antibodies in the serum samples of different species, with a high coincidence rate and no statistically significant difference compared with existing commercial ELISA kits. There was also no statistically significant difference for the developed cELISA and HI test. Compared with the commercial ELISA kits and HI test for detection of anti-IAV antibodies in different species, the method developed in the present study had obvious advantages regarding a simpler preparation process, lower cost, higher sensitivity, and shorter detection time, and thus has the potential to become a technology for the detection of IAVs in the future. Additionally, the platform of developing the cELISA based on the nanobody-HRP fusion is a novel and promising strategy for designing diagnostic kits for zoonotic disease, especially for pathogens with a wide range of potentially infectable hosts.
      The Influenza virus is a serious threat to human life and has caused global public health concerns (

      Webster, R. G., and Govorkova, E. A. J. A. o. t. N. Y. A. o. S. (2014) Continuing challenges in influenza. 1323

      ). As IAVs can infect a series of hosts including humans, poultry, pigs, minks, dogs, and other mammals, especially deep-sea mammals (
      • Webster R.G.
      • Bean W.J.
      • Gorman O.T.
      • Chambers T.M.
      • Kawaoka Y.
      Evolution and ecology of influenza A viruses.
      ), the development of a universal method for diagnosis in different species is important. The results of this study showed that the established cELISA could detect anti-IAV antibodies in humans, pigs, poultry, dogs, cattle, sheep, and other species. In addition, we also found that the epitope recognized by H9N2-NP-Nb5-HRP is highly conserved among all the different subtypes of IAVs. Therefore, we speculate that this method can be applied to the detection of anti-IAV antibodies in all species. Of course, in future studies, the more sera from different species will be assessed to verify the universality of the assay.
      It has been reported that AIV infections exist in the host of wild birds, resulting in the continuous spread of avian influenza across the world. Therefore, closer monitoring of AIV in wild birds is very important for better understanding the scope and transmission of infection (
      • Gulyaeva M.
      • De Marco M.A.
      • Kovalenko G.
      • Bortz E.
      • Murashkina T.
      • Yurchenko K.
      • Facchini M.
      • Delogu M.
      • Sobolev I.
      • Gadzhiev A.
      • Sharshov K.
      • Shestopalov A.
      Biological Properties and Genetic Characterization of Novel Low Pathogenic H7N3 Avian Influenza Viruses Isolated from Mallard Ducks in the Caspian Region, Dagestan, Russia.
      ). In the present study, the results showed that the established cELISA could detect anti-IAV antibodies in the sera from wild birds. As the number of wild birds used in the study was small, we will continue to collect the sera of different wild birds to prove the feasibility of the assay in detecting antibodies against AIV in wild birds. However, based on these data, it is hypothesized that the cELISA is a suitable means for monitoring the AIV infection in the wild bird flocks.
      To date, six epitopes have been determined in the NP protein from H5N1, H3N3, and H1N1 subtype IAVs (
      • Muñoz-Medina J.E.
      • Sánchez-Vallejo C.J.
      • Méndez-Tenorio A.
      • Monroy-Muñoz I.E.
      • Angeles-Martínez J.
      • Santos Coy-Arechavaleta A.
      • Santacruz-Tinoco C.E.
      • González-Ibarra J.
      • Anguiano-Hernández Y.-M.
      • González-Bonilla C.R.
      • Ramón-Gallegos E.
      • Díaz-Quiñonez J.A.
      In Silico Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential.
      ) (
      • Bui H.-H.
      • Peters B.
      • Assarsson E.
      • Mbawuike I.
      • Sette A.
      Ab and T cell epitopes of influenza A virus, knowledge and opportunities.
      ). However, all the above-identified epitopes were linear. In this study, we identified a conformational epitope of NP protein recognized by H9N2-NP-Nb5 and found that it is highly conserved in different subtypes of IAV. The epitope was predicted using the AlphaFold2 server, and certain regions for the reaction between H9N2-NP-Nb5-HRP and H9N2-NP were also determined using the different truncated and mutated fragments. However, the key amino acids have not been identified yet. In future studies, cryo-electron microscopy should be used to determine the spatial conformation of the antigen-nanobody complex and identify the key motifs. Additionally, our study showed that the epitope could induce strong immune responses in the different species after natural IAV infection. This suggested that the common epitope may serve an important function, which will be evaluated in future studies.
      In conclusion, a common and highly conserved epitope in the NP of different subtype IAVs recognized by the H9N2-NP-Nb5 was identified. Next, a cELISA using H9N2-NP-Nb5-HRP as a probe was developed and it exhibited good sensitivity, specificity, and high consistency with the commercial IDEXX ELISA kit. Importantly, the advantages of the developed cELISA over commercial kits include the elimination of enzyme-labeled antibodies in vitro, consistency between batches, and a simpler preparation process, lower cost, higher sensitivity and specificity, and shorter detection time (∼40 min in total). The developed cELISA was a suitable method for surveillance and monitoring of IAV infection in different species.

      Experimental procedures

      Cells and viruses

      Human-embryonic kidney 293T (HEK-293T) cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Inc., USA) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc., USA) at 37°C in a humidified incubator supplied with 5% CO2.
      A/chicken/Hebei/LC/2008 (H9N2 AIV, HB08 strain) was propagated in the allantoic cavity of 9-day-old chicken embryonated eggs. The inactivated and purified H5N1 and H7N9 viral particles were kindly provided by Harbin Veterinary Research Institute, CAAS, China.

      Serum samples

      The 180 negative sera from humans (n = 65), pigs (n = 60), and specific pathogen-free (SPF) chickens (Beijing Merial Vital Laboratory Animal Technology Co., Ltd, China, n = 55) were used to calculate the percentage inhibition (PI) of the developed cELISA. All these serum samples were confirmed using an IDEXX AIV antibody ELISA kit IDEXX (Westbrook, ME, USA) and HI assay. The commercial ELISA kit is the most stable and used in laboratory tests at present (
      • Du T.
      • Zhu G.
      • Wu X.
      • Fang J.
      • Zhou E.-M.
      Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.
      ,
      • Panyasing Y.
      • Goodell C.K.
      • Wang C.
      • Kittawornrat A.
      • Prickett J.R.
      • Schwartz K.J.
      • Ballagi A.
      • Lizano S.
      • Zimmerman J.J.
      Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA.
      ). And the HI assay is the serological "gold standard" method specified by OIE (
      • Watcharatanyatip K.
      • Boonmoh S.
      • Chaichoun K.
      • Songserm T.
      • Woratanti M.
      • Dharakul T.
      Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.
      ,
      • Shriner S.A.
      • VanDalen K.K.
      • Root J.J.
      • Sullivan H.J.
      Evaluation and optimization of a commercial blocking ELISA for detecting antibodies to influenza A virus for research and surveillance of mallards.
      ).
      In addition, to evaluate the agreements of the developed cELISA with the IDEXX commercial ELISA kit and with the HI assay, 91 sera collected from 13 SPF chickens challenged with H9N2 allantoic fluid (HA titer: 29) and 185 clinical sera were used for the simultaneous detection of three methods (
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ). To determine the cross-reactivity of the cELISA, cELISA was used to assess the 181 clinical positive chicken sera against other avian viruses, including Newcastle disease virus (NDV, n = 44), avian infectious bronchitis virus (IBV, n = 27), infectious bursal disease virus (IBDV, n = 33), avian leukemia virus (ALV, n = 23), Marek’s disease virus (MDV, n = 21), and avian hepatitis E virus (aHEV, n = 33), to assess the 116 clinical positive pig sera against other viruses, including porcine reproductive and respiratory syndrome virus (PRRSV, n = 23), porcine epidemic diarrhea virus (PEDV, n = 27), porcine circovirus type 2 (PCV2, n = 43), and pseudorabies virus (PRV, n = 23), and to assess the 69 positive human sera against other human viruses including hepatitis B Virus (HBV, n = 26), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, n = 15) and human adenovirus (HAdV, n = 21).

      Expression and purification of the recombinant H9N2-NP protein

      The H9N2 AIV strain HB08 was used as the template for amplification of the viral gene encoding the NP protein by reverse transcription-polymerase chain reaction (RT-PCR). The sequences of the primers used for PCR amplification are listed in Table S1. The gene was cloned into the pET-28a vector (Novagen, Germany) using the EcoR I and Xho I restriction enzyme sites. To express the recombinant H9N2-NP protein, the recombinant positive plasmids were transformed into E. coli Transetta (DE3) cells (TransGen Biotech, Beijing, China). Then, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for inducing the expression of recombinant H9N2-NP protein at 25°C for 12 h. After centrifugation at 8, 000 g for 15 min at 4°C, the bacteria were collected. After the bacterial cells were sonicated, the recombinant protein was purified using a Ni-NTA column (GE Healthcare, USA). Expression, purification, and antigenicity of the recombinant protein were analyzed by SDS-PAGE and western blotting.

      Bactrian camel immunization and library construction

      A 4-year-old male Bactrian camel was immunized with the purified recombinant H9N2-NP protein (1 mg/mL) with an equal volume of complete Freund’s adjuvant (Sigma-Aldrich; Merck KGaA, St. Louis, MO, US) subcutaneously (
      • Du T.
      • Zhu G.
      • Wu X.
      • Fang J.
      • Zhou E.-M.
      Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.
      ,
      • Lu Q.
      • Li X.
      • Zhao J.
      • Zhu J.
      • Luo Y.
      • Duan H.
      • Ji P.
      • Wang K.
      • Liu B.
      • Wang X.
      • Fan W.
      • Sun Y.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays.
      ). Five consecutive immunizations were performed every 2 weeks. For the subsequent four administrations, the recombinant proteins were emulsified with incomplete Freund's adjuvant and the injection volume was the same as that used the first time. After the last immunization, the sera were collected from the immunized camel and used to test the titer of anti-H9N2-NP antibodies with indirect ELISA (iELISA) using purified H9N2-NP protein as a coating antigen.
      A total of 5 days after the last immunization, 200 mL of peripheral blood was collected from the immunized Bactrian camel and diluted with an equal volume of RPMI 1640 media (01-100-1ACS, Biological Industries, Kibbutz Beit Haemek, Israel). Next, the Ficoll-Paque PLUS Lymphocyte Separation Fluid (Greiner bio-one, Germany) was used to isolate peripheral blood lymphocytes (PBLs) according to the manufacturer’s protocol. Total RNA was extracted from the PBLs and used as templates for reverse transcription to synthesize the cDNA. Next, the VHH genes were amplified using nested PCR as described previously (
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ,
      • Liu H.
      • Wang Y.
      • Duan H.
      • Zhang A.
      • Liang C.
      • Gao J.
      • Zhang C.
      • Huang B.
      • Li Q.
      • Li N.
      • Xiao S.
      • Zhou E.-M.
      An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication.
      ,
      • Ji P.
      • Zhu J.
      • Li X.
      • Fan W.
      • Liu Q.
      • Wang K.
      • Zhao J.
      • Sun Y.
      • Liu B.
      • Zhou E.-M.
      • Zhao Q.
      Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus.
      ). The sequences of the primers used for nested PCR are listed in Table 1. Subsequently, the VHH genes were cloned into a phage display pMECS vector using the Pst I and Not I restriction enzyme sites. The recombinant ‘phagemids’ were electroporated into 1 mL fresh E. coli TG1 competent cells. The cells were plated on Luria-Bertani (LB) agar plates supplemented with ampicillin and D-glucose at 37°C. The bacterial monolayer was scraped and added to LB containing 20% glycerin and stored at -80°C. Then, the positive rate and diversity of the library were determined as described previously (
      • Vincke C.
      • Gutierrez C.
      • Wernery U.
      • Devoogdt N.
      • Hassanzadeh-Ghassabeh G.
      • Muyldermans S.
      Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.
      ).

      Screening of anti-H9N2-NP-specific nanobodies.

      To screen the specific nanobodies against the H9N2-NP protein, three rounds of panning were performed as previously described (
      • Liu H.
      • Wang Y.
      • Duan H.
      • Zhang A.
      • Liang C.
      • Gao J.
      • Zhang C.
      • Huang B.
      • Li Q.
      • Li N.
      • Xiao S.
      • Zhou E.-M.
      An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication.
      ,
      • Ji P.
      • Zhu J.
      • Li X.
      • Fan W.
      • Liu Q.
      • Wang K.
      • Zhao J.
      • Sun Y.
      • Liu B.
      • Zhou E.-M.
      • Zhao Q.
      Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus.
      ). Briefly, the 96-well plates (Nunc, Thermo Fisher Scientific, Inc.) were coated with the purified recombinant H9N2-NP protein (5 μg/well) using phosphate buffer (PBS, pH 7.2) as the coating buffer. Then, the blocking buffer (2.5% skimmed milk in PBS, 300 μL/well) was added to the plates at 37°C for 2 h. After washing with PBS’T (1 L PBS with 1 mL Tween 20, 300 μL/well), the recombinant phages rescued via M13K07 (5 × 1011 PFU/mL) were added and incubated for 2 h at room temperature (RT). Then, the binding phages were eluted with fresh trimethylamine (100 mM) and neutralized with Tris-HCl buffer (1 M, pH 7.4). The titers of eluted phages were tested by infecting TG1 cells, and then used for the next round of rescue and panning. After three rounds of screening, the ratio of the output of the positive phage to the output of the negative phage was calculated to evaluate the enrichment of phages against H9N2-NP. A total of 96 clones were selected and induced by 1 mM IPTG for expression of anti-H9N2-NP nanobodies. The soluble nanobodies were extracted from the periplasm and tested by iELISA using the H9N2-NP protein as the coating antigen. The NDV-NP was used as a negative control for the iELISA. The protein was also expressed using the same vector pET-28a described in a previous study (
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ). Finally, all VHH genes from the positive clones were sequenced (Tsingke Biotechnology Co., Ltd, Beijing, China) and classified according to their complementary determining regions (CDRs) amino acid sequence.

      iELISA

      For determining the titers of immunized Bactrian camel and specific detection of the periplasm containing the nanobodies, iELISA was performed based on a previous study (
      • Ji P.
      • Zhu J.
      • Li X.
      • Fan W.
      • Liu Q.
      • Wang K.
      • Zhao J.
      • Sun Y.
      • Liu B.
      • Zhou E.-M.
      • Zhao Q.
      Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus.
      ). Briefly, the 96-well plates were coated with the purified H9N2-NP (400 ng/well) overnight at 4°C and then were blocked with blocking buffer at 37°C for 1 h. After the plates were washed three times with PBS’T, the sera from the immunized camel or the periplasm were added to the wells and incubated at RT for 1 h. After washing three times, for titration of camel sera, the rabbit anti-camel antibody and HRP-labeled goat anti-rabbit IgG (TransGen Biotech, China) were added sequentially. For testing the periplasm, the anti-HA-Tag antibody (GenScript Biotech Corp, China) and HRP-labeled goat anti-mouse IgG (TransGen Biotech, China) were subsequently added. After washing again as above, tetramethylbenzidine (TMB) was added to the plates and the plates were incubated at RT for 15 min to allow the development of the color. Subsequently, the reaction was halted by the addition of 3 M H2SO4, and the OD450nm value was read using an automatic ELISA plate reader (Bio-Rad Laboratories, Inc., USA).

      Expression of the anti-H9N2-NP nanobody with HRP fusion proteins.

      The nanobody-HRP fusion protein was expressed in HEK-293T cells as described previously (

      Yamagata, M., and Sanes, J. R. (2018) Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. 201722491

      ). Briefly, the VHH genes encoding the nanobody were obtained from the positive pMECS plasmids by digesting with Pst I and Not I enzymes and ligating into the vector pCMV-N1-HRP, which was also digested with the same two enzymes, as described previously (
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ). After the positive recombinant plasmids were identified by sequencing, they were transfected into the HEK-293T cells with polyetherimide (PEI, Polysciences Inc. Warrington, USA) agent. A total of 48 h post-transfection, the culture supernatant containing the nanobody-HRP fusion proteins were collected and supplemented with 0.02% w/v NaN3. For identification of expression, the transfected cells were analyzed using an indirect immunofluorescence assay (IFA) with anti-His monoclonal antibodies as the primary antibody and FITC labeled goat anti-mice IgG antibodies as the second antibody. Direct ELISA was performed to quantify the secreted expression of nanobody-HRP fusion proteins in the HEK-293T cells as described previously (
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ). Briefly, after the plate had been coated with the purified H9N2-NP protein, the culture medium (100 μL/well) was added and incubated for 1 h at RT. As above, the TMB color reactions were halted with 3 M H2SO4 and OD450nm value was obtained.

      Cross-reaction of the nanobody-HRP fusion protein with other IAV subtypes

      Cross-reactions of these nanobody-HRP fusion proteins with other subtypes of IAVs were also assessed using the direct ELISA described above. The A/Guangzhou/01/2009 (H1N1), A/swine/Guangxi/1659/2017 (H3N2), H5N1 (Re-12), H7N9 (H7-Re3) IAV strains and influenza B virus (IBV) strain (B/Yamagata/16/1988) were selected for use as coating antigens. Of these, the H1N1, H3N2 IAVs NPs and IBV NP were also expressed using the bacterial system as for the H9N2 NP and used as coating antigens in the direct ELISA. For H5N1 and H7N9, the purified and denatured viral particles were used as coating antigens.
      In addition, to further analyze cross-reaction, the blocking rate of the nanobody-HRP fusion proteins blocked by the positive sera for antibodies against different subtypes of IAVs to react with the antigens was also determined using the blocking ELISA as described previously (
      • Sheng Y.
      • Wang K.
      • Lu Q.
      • Ji P.
      • Liu B.
      • Zhu J.
      • Liu Q.
      • Sun Y.
      • Zhang J.
      • Zhou E.-M.
      • Zhao Q.
      Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
      ). Briefly, the ELISA plates were coated with the purified H9N2-NP protein (200 ng/well) at 4°C overnight. Then, the positive and negative sera for anti-H9N2, H5N1, H7N9, H1N1, and H3N2 antibodies were added to the wells and incubated at RT for 1 h. After washing with PBS’T, the same amount of medium containing the nanobody-HRP fusion proteins (100 μL/well) was added to each well. After the OD450nm values were obtained, the blocking rates were calculated using the following forum: [1 - (OD450nm value of positive sera/OD450nm value of negative sera)] x 100%. Then, the nanobody-HRP fusion protein being cross-reacted with other subtypes of IAVs and with the largest blocking rate was selected for identification of recognizing epitopes and as a probe to develop the cELISA for detecting anti-IAV antibodies in different species.

      Identification of the epitope recognized by the nanobody-HRP fusion protein

      To determine the epitope recognized by the selected nanobody-HRP fusion protein, a series of truncated and overlapping fragments from H9N2-NP were designed and expressed within the bacterial system. The H9N2-NP protein was first divided into two equal fragments and then, the two fragments were extended by 1/4 and 1/8 to the N-terminal and C-terminal, respectively (Figure 4A). Based on the nucleotide sequences encoding H9N2-NP, primer pairs were designed for amplifying these fragments (Table S1). The expression and purification procedures of truncated fragments were the same as that for the full-length H9N2-NP. SDS-PAGE and western blotting assays were used to analyze the expression and purification of these truncated fragments. Using these purified fragments as coating antigens, the antigenic domain recognized by the nanobody-HRP fusion protein was determined by direct ELISA.
      To define the key amino acids involved in the interaction between the nanobody-HRP fusion protein and H9N2-NP protein more precisely, the 3D structures of homology modeling for H9N2-NP and the nanobody were generated by submitting the amino acid sequences of the two proteins to the AlphaFold2 server (
      • Jumper J.
      • Evans R.
      • Pritzel A.
      • Green T.
      • Figurnov M.
      • Ronneberger O.
      • Tunyasuvunakool K.
      • Bates R.
      • Zidek A.
      • Potapenko A.
      • Bridgland A.
      • Meyer C.
      • Kohl S.A.A.
      • Ballard A.J.
      • Cowie A.
      • Romera-Paredes B.
      • Nikolov S.
      • Jain R.
      • Adler J.
      • Back T.
      • Petersen S.
      • Reiman D.
      • Clancy E.
      • Zielinski M.
      • Steinegger M.
      • Pacholska M.
      • Berghammer T.
      • Bodenstein S.
      • Silver D.
      • Vinyals O.
      • Senior A.W.
      • Kavukcuoglu K.
      • Kohli P.
      • Hassabis D.
      Highly accurate protein structure prediction with AlphaFold.
      ). The docking model of the interactions of the two proteins was then developed using the docking program on the server ClusPro (cluspro.bu.edu/home.php) (
      • Kozakov D.
      • Hall D.R.
      • Xia B.
      • Porter K.A.
      • Padhorny D.
      • Yueh C.
      • Beglov D.
      • Vajda S.
      The ClusPro web server for protein-protein docking.
      ). Interaction sites were analyzed using PyMOL (pymol.org/2/support.html) (
      • Kagami L.P.
      • das Neves G.M.
      • Timmers L.F.S.M.
      • Caceres R.A.
      • Eifler-Lima V.L.
      Geo-Measures: A PyMOL plugin for protein structure ensembles analysis.
      ).
      Subsequently, the predicted key amino acids were separately mutated to Alanine (A) residues for H9N2-NP and the nanobody to identify the predicted results. The primers used for PCR amplification of the mutated fragments are listed in Table 1. The mutated H9N2-NP protein and nanobody-HRP fusion protein were expressed using the bacterial system and HEK293T cells, respectively. Finally, direct ELISA using mutated H9N2-NP as the coating antigen and the nanobody-HRP fusion protein as the testing antibody was performed to confirm the predicted results.

      Amino acid alignment of the epitopes recognized by the nanobody-HRP fusion protein

      To analyze the conservation of the epitope among the different subtype IAV strains, the amino acid sequences of the antigenic domains from different strains in GenBank were aligned using the Clustal W module of Lasergene 7.1 (DNASTAR, MegAlign). The GenBank accession numbers of different IAV strains were A/chicken/Hebei/0822/2007 (H9N2) (GQ373096.1), A/California/04/2009 (H1N1) (MN371617.1), A/swine/Guangxi/JG1/2014 (H3N2) (MF927881.1), A/HK/212/03 (H5N1) (AY575905.1), A/tree sparrow/Shanghai/01/2013 (H7N9) (KF609528.1), A/swine/Gunma/1/2012 (H1N2) (AB731586.1), A/Berkeley/1/68 (H2N2) (AY210104.1), A/canine/Florida/242/2003 (H3N8) (DQ124158.1), A/mallard/California/57871/2015 (H4N6) (KX351682.1), A/mallard/Sweden/100993/2008 (H7N7) (FJ803195.2), A/swan/Shandong/W4322/2020 (H10N4) (OM373303.1), A/mallard/Beijing/27-MA/2011 (H10N7) (KY688104.1), A/mallard/Korea/SH38-45/2010 (H13N2) (JX030409.1), and A/seagull/Chile/5775/2009 (H13N9) (KF772957.1).

      Development of the cELISA for detecting anti-IAV antibodies

      Using the selected nanobody-HRP fusion protein as the probe, the cELISA for detecting anti-IAV antibodies in serum samples was designed as described previously (
      • Du T.
      • Zhu G.
      • Wu X.
      • Fang J.
      • Zhou E.-M.
      Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.
      ). First, a checkerboard titration using direct ELISA was used to determine the optimal amount of coating antigen and dilution of the selected nanobody-HRP fusion protein. The amounts of coated H9N2-NP protein assessed were 50, 100, 200, and 400 ng/well, and the dilution ratios of the nanobody-HRP fusion assessed were 1:320, 1:640, 1:1,280, 1:2,000, 1:4,000, and 1:6,000 separately used for the direct ELISA. The optimized amount of antigen and dilution of nanobody-HRP fusion were determined when the pairs in the direct ELISA produced an OD450nm value of ∼1.0.
      Second, the dilutions of testing sera were optimized for the cELISA. The different dilutions (1:10, 1:20, 1:40, and 1:80) of five positive (P) and negative (N) sera for anti-IAV antibodies (each serum sample of H9N2, H1N1, H3N2, and H5N1, H7N9) were separately mixed with the optimized dilution of the nanobody-HRP fusion protein. After the optimized amount of H9N2-NP protein was coated into the plate, the mixtures were added to the plate and incubated for 1 h at 37°C. Then, the TMB was added and the OD450nm values were read after the reaction was terminated. The optimal dilution of testing chicken sera was determined when the ratio of the positive and the negative sera OD450nm values (P/N) was the smallest.
      Third, the incubation times between the mixture, the coated antigen, and the color reaction times after adding TMB were also optimized using the checkerboard assay. The incubation times of 20, 30, 40, 50, and 60 min and the color reaction times of 10 and 15 min were selected. Then, the two optimal times were determined when the P/N value was the smallest.
      After the above conditions were optimized, the developed cELISA was performed as followed. The 96-well ELISA plates were coated with the optimized amount of H9N2-NP protein in PBS (0.1 M, pH 7.4) at 4°C overnight. Then, the plates were washed and blocked with 300 μL/well blocking buffer (2.5% milk in PBS’T) at 37°C for 1 h. After washing with PBS’T, 100 μL testing mixture containing the optimal dilutions of serum sample and nanobody-HRP fusion was added to wells and incubated for the optimal time at 37°C. After washing three times, 100 μL TMB was added and incubated for the optimal time at RT, and then the OD450nm values were measured after the reaction had been stopped by 3 M H2SO4.

      Validation of the cELISA

      To calculate the percentage inhibition (PI) of the cELISA, the 180 sera samples negative for anti-IAV antibodies from humans, pigs, and chicken were used. The cut-off value was set to the average PI of 180 negative sera plus 3 standard deviations (SD) to ensure 99% confidence that the negative sera was within this range.
      The sensitivity of cELISA was evaluated by testing the different dilutions (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280, and 1:2,560) of 48 positive sera for anti-H9N2 (n = 16), H5N1 (n = 8), H7N9 (n = 8), H1N1 (n = 8), and H3N2 (n = 8) antibodies.
      To determine the specificity of cELISA, a total of 181 clinical positive chicken sera against other avian viruses including NDV (n = 44), IBV (n = 27), IBDV (n = 33), ALV (n = 23), MDV (n = 21), and aHEV (n = 33), 128 positive pig sera against other swine viruses including PRRSV (n = 23), PEDV (n = 27), PCV ( n = 43), and PRV (n = 23), and 69 positive human sera against other human viruses including HBV (n = 26), SARS-CoV-2 (n = 15), and HAdV (n = 21) were used to test the developed cELISA.
      The reproducibility of the developed cELISA was analyzed by testing five positive and negative sera to analyze the intra-assay and inter-assay variabilities. The coefficient of variation (CV) was calculated using the PI values of different sera to evaluate the inter-assay variation (between plates) and the intra-assay variation (within a plate). Each serum sample was tested using three different plates to determine the inter-assay CV and three replicates within each plate to calculate the intra-assay CV.
      The stability of the nanobody-HRP fusion and the developed cELISA were also analyzed. Briefly, the 96-well plates were coated with the purified recombinant H9N2-NP protein at 4°C for 12 h. After washing with PBS’T and blocking with the blocking buffer, the plates were dried in a fume hood and vacuumed. Then, the plates and optimized dilutions of the selected nanobody-HRP fusion were stored at 4°C. The direct ELISA and cELISA were separately performed as described above after 0, 30, 60, 90, 120, 150, and 180 days to evaluate the stability.

      Analysis of the cELISA to detect anti-IAV antibodies in different species

      To evaluate the developed cELISA for the detection of anti-IAV antibodies in the sera from different species, the 2,155 sera from humans (n = 660), swine (n = 660), goats (n = 195), cattle (n = 28), rabbits (n = 44), cats (n = 44), pet dogs (n = 524), ducks (n = 98), and wild birds (n = 63) were both tested with the developed assay and an IDEXX IAV antibody ELISA kit (IDEXX, Westbrook, ME, USA).

      Comparisons of the analytical performance among the cELISA, commercial ELISA kits, and HI test

      Reference methods for detection of anti-IAV antibody in laboratory and literature include HI test, AGID, VNT, IFA, and ELISA (
      • Saczynska V.
      • Florys-Jankowska K.
      • Porebska A.
      • Cecuda-Adamczewska V.
      A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin.
      ,
      • Li Y.
      • Ye H.
      • Liu M.
      • Song S.
      • Chen J.
      • Cheng W.
      • Yan L.
      Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus.
      ). Among them, the HI assay is the serological "gold standard" method by OIE (
      • Watcharatanyatip K.
      • Boonmoh S.
      • Chaichoun K.
      • Songserm T.
      • Woratanti M.
      • Dharakul T.
      Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.
      ,
      • Shriner S.A.
      • VanDalen K.K.
      • Root J.J.
      • Sullivan H.J.
      Evaluation and optimization of a commercial blocking ELISA for detecting antibodies to influenza A virus for research and surveillance of mallards.
      ). Additionally, the ELISA is also widely used for testing anti-IAV antibody in the laboratory. Among these available commercial ELISA kits, the commercial IDEXX ELISA kit is currently the most stable and used for detection of anti-IAV antibodies. Therefore, the IDEXX ELISA kit and HI test were performed on a comparison of the analytical performance of the developed cELISA in this study.
      The 185 clinical chicken sera and 91 sequential sera from 13 SPF chickens challenged with H9N2 AIV stock were simultaneously tested using the developed cELISA, a commercial IDEXX ELISA kit, and the HI test. Then, the coincidence rates were also calculated using Microsoft Excel’s CORREL function.

      Statistical analysis

      Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). The κ index values were calculated to estimate the coincidence between the developed cELISA and the commercial ELISA kit (

      Langley, R. J. J. O. A. (1996) Veterinary Clinical Epidemiology: A Problem Oriented Approach, by Ronald Smith. 3, 63-64

      ). These calculations were performed using SPSS version 20 (IBM Corp.).

      Data availability

      All data are provided within the article.

      Acknowledgments

      We are very grateful to researcher Wang Xiurong of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences for the gift of the H5N1 and H7N9 inactivated viruses.

      References

        • Cox N.J.,S.K.
        Influenza : The Lancet. 1999; 354: 1277-1282
        • Wille M.
        • Holmes E.C.
        The Ecology and Evolution of Influenza Viruses.
        Cold Spring Harb Perspect Med. 2020; 10
        • Webster R.G.
        • Govorkova E.A.
        Continuing challenges in influenza.
        Ann N Y Acad Sci. 2014; 1323: 115-139
      1. Palese, P., and Young, J. F. (1982) Variation of Influenza A, B, and C Viruses. 215, 1468-1474

        • Saczynska V.
        • Florys-Jankowska K.
        • Porebska A.
        • Cecuda-Adamczewska V.
        A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin.
        Virol J. 2021; 18: 91
        • Webster R.G.
        • Bean W.J.
        • Gorman O.T.
        • Chambers T.M.
        • Kawaoka Y.
        Evolution and ecology of influenza A viruses.
        Microbiol Rev. 1992; 56: 152-179
      2. Animals, W. O. f. A. H. M. o. D. T. a. V. f. T. (2018) Chapter 3.3.4. Avian influenza (infection with avian influenza viruses).

        • Nobusawa E.
        • Aoyama T.
        • Kato H.
        • Suzuki Y.
        • Tateno Y.
        • Nakajima K.
        Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses.
        Virology. 1991; 182: 475-485
      3. Sreenivasan, C. C., Thomas, M., Kaushik, R. S., Wang, D., and Li, F. (2019) Influenza A in Bovine Species: A Narrative Literature Review. Viruses 11

        • Matrosovich M.N.
        • Matrosovich T.Y.
        • Gray T.
        • Roberts N.A.
        • Klenk H.-D.
        Human and avian influenza viruses target different cell types in cultures of human airway epithelium.
        Proceedings of the National Academy of Sciences of the United States of America. 2004; 101: 4620-4624
        • Hay A.J.
        • Gregory V.
        • Douglas A.R.
        • Lin Y.P.
        The evolution of human influenza viruses.
        Philos Trans R Soc Lond B Biol Sci. 2001; 356: 1861-1870
        • Abolnik C.
        • Fehrsen J.
        • Olivier A.
        • van Wyngaardt W.
        • Fosgate G.
        • Ellis C.
        Serological investigation of highly pathogenic avian influenza H5N2 in ostriches (Struthio camelus).
        Avian Pathol. 2013; 42: 206-214
        • Li Y.
        • Hu J.
        • Lei J.
        • Fan W.
        • Bi Z.
        • Song S.
        • Yan L.
        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. 2021; 166: 1113-1124
        • Li Y.
        • Ye H.
        • Liu M.
        • Song S.
        • Chen J.
        • Cheng W.
        • Yan L.
        Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus.
        BMC Vet Res. 2021; 17: 64
        • Honda T.
        • Gomi S.
        • Yamane D.
        • Yasui F.
        • Yamamoto T.
        • Munakata T.
        • Itoh Y.
        • Ogasawara K.
        • Sanada T.
        • Yamaji K.
        • Yasutomi Y.
        • Tsukiyama-Kohara K.
        • Kohara M.
        Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies.
        mSphere. 2021; 6
        • Yang M.
        • Berhane Y.
        • Salo T.
        • Li M.
        • Hole K.
        • Clavijo A.
        Development and application of monoclonal antibodies against avian influenza virus nucleoprotein.
        Journal of Virological Methods. 2008; 147: 265-274
      4. Shu, L. L., Bean, W. J., and Webster, R. G. J. J. o. V. (1993) Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. 67, 2723-2729

        • Tu Y.-C.
        • Chen K.-Y.
        • Chen C.-K.
        • Cheng M.-C.
        • Lee S.-H.
        • Cheng I.-C.
        Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying.
        Journal of veterinary science. 2019; 20: 51-57
        • Hu Y.
        • Sneyd H.
        • Dekant R.
        • Wang J.
        Influenza A Virus Nucleoprotein: A Highly Conserved Multi-Functional Viral Protein as a Hot Antiviral Drug Target.
        Curr Top Med Chem. 2017; 17: 2271-2285
        • Du T.
        • Zhu G.
        • Wu X.
        • Fang J.
        • Zhou E.-M.
        Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.
        International journal of nanomedicine. 2019; 14: 9337-9349
        • Goodell C.K.
        • Prickett J.
        • Kittawornrat A.
        • Johnson J.
        • Zhang J.
        • Wang C.
        • Zimmerman J.J.
        Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine.
        Transboundary and Emerging Diseases. 2016; 63: 24-35
        • Muyldermans S.
        • Lauwereys M.
        Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies.
        Journal of Molecular Recognition. 1999; 12: 131-140
        • Li D.
        • Ji F.
        • Huang C.
        • Jia L.
        High Expression Achievement of Active and Robust Anti-β2 microglobulin Nanobodies via E.coli Hosts Selection.
        Molecules. 2019; 24: 2860
        • Wrapp D.
        • De Vlieger D.
        • Corbett K.S.
        • Torres G.M.
        • Wang N.
        • Van Breedam W.
        • Roose K.
        • van Schie L.
        • Team V.-C.C.-R.
        • Hoffmann M.
        • Pohlmann S.
        • Graham B.S.
        • Callewaert N.
        • Schepens B.
        • Saelens X.
        • McLellan J.S.
        Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies.
        Cell. 2020; 181 (1015 e1015): 1004
        • Ren W.
        • Li Z.
        • Xu Y.
        • Wan D.
        • Barnych B.
        • Li Y.
        • Tu Z.
        • He Q.
        • Fu J.
        • Hammock B.D.
        One-Step Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Fusion for Detection of Aflatoxin B(1) in Cereal.
        J Agric Food Chem. 2019; 67: 5221-5229
        • Lu Q.
        • Li X.
        • Zhao J.
        • Zhu J.
        • Luo Y.
        • Duan H.
        • Ji P.
        • Wang K.
        • Liu B.
        • Wang X.
        • Fan W.
        • Sun Y.
        • Zhou E.-M.
        • Zhao Q.
        Nanobody-horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays.
        Journal of nanobiotechnology. 2020; 18 (7): 7
        • Sheng Y.
        • Wang K.
        • Lu Q.
        • Ji P.
        • Liu B.
        • Zhu J.
        • Liu Q.
        • Sun Y.
        • Zhang J.
        • Zhou E.-M.
        • Zhao Q.
        Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.
        Journal of nanobiotechnology. 2019; 17 (35): 35
        • Panyasing Y.
        • Goodell C.K.
        • Wang C.
        • Kittawornrat A.
        • Prickett J.R.
        • Schwartz K.J.
        • Ballagi A.
        • Lizano S.
        • Zimmerman J.J.
        Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA.
        Transboundary and Emerging Diseases. 2014; 61: 177-184
        • Watcharatanyatip K.
        • Boonmoh S.
        • Chaichoun K.
        • Songserm T.
        • Woratanti M.
        • Dharakul T.
        Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.
        Journal of Virological Methods. 2010; 163: 238-243
        • Gao Y.
        • Huang X.
        • Zhu Y.
        • Lv Z.
        A brief review of monoclonal antibody technology and its representative applications in immunoassays.
        Journal of Immunoassay and Immunochemistry. 2018; 39: 351-364
        • Lequin R.M.
        Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA).
        Clinical Chemistry. 2005; 51: 2415-2418
      5. Duan, H., Chen, X., Zhao, J., Zhu, J., Zhang, G., Fan, M., Zhang, B., Wang, X., Sun, Y., Liu, B., Zhou, E.-M., Zhao, Q., and Theel Elitza, S. Development of a Nanobody-Based Competitive Enzyme-Linked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses. Journal of clinical microbiology 59, e01580-01521

        • Kumar V.
        • Mishra B.
        Monoclonal antibodies and its applications: An overview.
        Acta Pharmaceutica Turcica. 1999; XLI: 191-198
        • Pillay T.S.
        • Muyldermans S.
        Application of Single-Domain Antibodies ("Nanobodies") to Laboratory Diagnosis.
        Ann Lab Med. 2021; 41: 549-558
      6. Webster, R. G., and Govorkova, E. A. J. A. o. t. N. Y. A. o. S. (2014) Continuing challenges in influenza. 1323

        • Gulyaeva M.
        • De Marco M.A.
        • Kovalenko G.
        • Bortz E.
        • Murashkina T.
        • Yurchenko K.
        • Facchini M.
        • Delogu M.
        • Sobolev I.
        • Gadzhiev A.
        • Sharshov K.
        • Shestopalov A.
        Biological Properties and Genetic Characterization of Novel Low Pathogenic H7N3 Avian Influenza Viruses Isolated from Mallard Ducks in the Caspian Region, Dagestan, Russia.
        Microorganisms. 2021; 9: 864
        • Muñoz-Medina J.E.
        • Sánchez-Vallejo C.J.
        • Méndez-Tenorio A.
        • Monroy-Muñoz I.E.
        • Angeles-Martínez J.
        • Santos Coy-Arechavaleta A.
        • Santacruz-Tinoco C.E.
        • González-Ibarra J.
        • Anguiano-Hernández Y.-M.
        • González-Bonilla C.R.
        • Ramón-Gallegos E.
        • Díaz-Quiñonez J.A.
        In Silico Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential.
        BioMed research international 2015. 2015; (813047): 813047
        • Bui H.-H.
        • Peters B.
        • Assarsson E.
        • Mbawuike I.
        • Sette A.
        Ab and T cell epitopes of influenza A virus, knowledge and opportunities.
        Proceedings of the National Academy of Sciences of the United States of America. 2007; 104: 246-251
        • Shriner S.A.
        • VanDalen K.K.
        • Root J.J.
        • Sullivan H.J.
        Evaluation and optimization of a commercial blocking ELISA for detecting antibodies to influenza A virus for research and surveillance of mallards.
        Journal of Virological Methods. 2016; 228: 130-134
        • Liu H.
        • Wang Y.
        • Duan H.
        • Zhang A.
        • Liang C.
        • Gao J.
        • Zhang C.
        • Huang B.
        • Li Q.
        • Li N.
        • Xiao S.
        • Zhou E.-M.
        An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication.
        Veterinary Microbiology. 2015; 181: 252-260
        • Ji P.
        • Zhu J.
        • Li X.
        • Fan W.
        • Liu Q.
        • Wang K.
        • Zhao J.
        • Sun Y.
        • Liu B.
        • Zhou E.-M.
        • Zhao Q.
        Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus.
        Journal of nanobiotechnology. 2020; 18 (44): 44
        • Vincke C.
        • Gutierrez C.
        • Wernery U.
        • Devoogdt N.
        • Hassanzadeh-Ghassabeh G.
        • Muyldermans S.
        Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.
        Methods Mol Biol. 2012; 907: 145-176
      7. Yamagata, M., and Sanes, J. R. (2018) Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. 201722491

        • Jumper J.
        • Evans R.
        • Pritzel A.
        • Green T.
        • Figurnov M.
        • Ronneberger O.
        • Tunyasuvunakool K.
        • Bates R.
        • Zidek A.
        • Potapenko A.
        • Bridgland A.
        • Meyer C.
        • Kohl S.A.A.
        • Ballard A.J.
        • Cowie A.
        • Romera-Paredes B.
        • Nikolov S.
        • Jain R.
        • Adler J.
        • Back T.
        • Petersen S.
        • Reiman D.
        • Clancy E.
        • Zielinski M.
        • Steinegger M.
        • Pacholska M.
        • Berghammer T.
        • Bodenstein S.
        • Silver D.
        • Vinyals O.
        • Senior A.W.
        • Kavukcuoglu K.
        • Kohli P.
        • Hassabis D.
        Highly accurate protein structure prediction with AlphaFold.
        Nature. 2021; 596: 583-589
        • Kozakov D.
        • Hall D.R.
        • Xia B.
        • Porter K.A.
        • Padhorny D.
        • Yueh C.
        • Beglov D.
        • Vajda S.
        The ClusPro web server for protein-protein docking.
        Nat Protoc. 2017; 12: 255-278
        • Kagami L.P.
        • das Neves G.M.
        • Timmers L.F.S.M.
        • Caceres R.A.
        • Eifler-Lima V.L.
        Geo-Measures: A PyMOL plugin for protein structure ensembles analysis.
        Computational Biology and Chemistry. 2020; 87107322
      8. Langley, R. J. J. O. A. (1996) Veterinary Clinical Epidemiology: A Problem Oriented Approach, by Ronald Smith. 3, 63-64