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

Abstract

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

On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a

“…And they also developed a H5 subtype AIVs assay based on CRISPR/Cas13a, RT-RAA, and LFD with a sensitivity of up to 0.1 copies [42]. In addition, S ączy ńska V et al established a novel epitope-blocking ELISA (EB-ELISA) for the specific and sensitive detection of anti-HA antibodies against the H5 subtype [43]. The sensitivity and specificity of the assay established in this study are comparable to those described above, but the method has advantages in terms of diversified forms of presentation, time-saving, and not strictly relying on specialized instruments.…”

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3

“…Antibodies specific to the virus in the serum bind to the pre-coated antigen, and this binding blocks the mAb from binding to the antigen, so the blocking ELISA has good specificity. This method has been widely used for the accurate and specific serological diagnosis of various diseases [39][40][41]. In this study, we innovatively established an EB-ELISA method for PCV3 antibody detection using the obtained mAb.…”

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA

“…In recent years, more and more detection methods have been applied to the detection of avian influenza virus, among which the isolation and identification of the virus is the gold standard for detection [ 28 ], but it needs to be carried out in the tertiary biosafety laboratory. Violetta Szczynska et al [ 28 ] established an ELISA based avian influenza virus detection method, which can effectively detect avian influenza virus. Hua Bai et al [ 29 ] established a surface plasma biosensor for detecting avian influenza virus.…”