A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin

Violetta Sączyńska,Katarzyna Florys-Jankowska,Violetta Cecuda-Adamczewska,Anna Porębska

doi:10.1186/s12985-021-01564-6

Abstract

BackgroundH5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA).MethodsIn the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1–H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis.ResultsThe H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively.ConclusionsThe H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

Highlights

H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic poten‐ tial
H5-subtype HPAIVs and LPAIVs with a propensity to mutate into HP viruses are under epidemiologic surveillance as Notifiable avian influenza virus (NAIV), which is accomplished using a variety of methods recommended by Organization for Animal Health (OIE) [3]
The H5 EB-enzymelinked immunosorbent assay (ELISA) developed in this study performed well in terms of analytical specificity (Asp), diagnostic specificity (Dsp) and Diagnostic sensitivity (Dse) when applied to screen chicken sera for the presence of H5 subtype-specific antibodies

Summary

Introduction

H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic poten‐ tial. Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. Influenza A viruses have antigenically related NP and M1 proteins [3]. They are classified into subtypes on the basis of their variable HA and NA antigens. Avian influenza viruses (AIVs) are usually nonpathogenic in their natural waterfowl hosts, and most of them are of low pathogenicity (LP) for domestic birds. The H5- and H7-subtype AIVs may become highly pathogenic (HP) by mutation once introduced into the poultry population

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