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A Novel Epitope-blocking ELISA for Specific and Sensitive Detection of Antibodies against H5-Subtype Influenza Virus Hemagglutinin
Violetta Sączyńska
Institute of Biotechnology and Antibiotics, Warsaw, Poland
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1828-6783
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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.
Keywords
Avian influenza viruses, Hemagglutinin, Monoclonal antibodies, Antibody detection, Blocking ELISA, H5, Serology
Figure 1
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile108.01.21.pdf
Additional file 1: Supplementary data on antigens and sera. Table S1. Recombinant H5 HA proteins (ITC, OET Ltd., IBA). Table S2. Reference antisera against LPAIVs of the H5 subtype (x-OvO Ltd.). Table S3. Reference antisera against LPAIVs of the non-H5 subtypes (x-OvO Ltd.). Table S4. Experimental antisera against HA from H5N1 HPAIV (IBA). Table S5. Antigens and antisera (x-OvO Ltd.) used in the HI tests performed at IBA. Table S6. Homology of H5 HA antigens against rH5-mammalian, an immunogen used in mAb production. Table S7. Homology of H5 HA antigens against rH5-BEVS, the coating antigen in the EB-ELISA.
- Additionalfile208.01.21.pdf
Additional file 2: Verification of mAb activity in the hemagglutination inhibition assay. Table S1. Antigens and antisera used in testing the mAbs for HI activity. Table S2. Commercial mAbs against H5 HA used as controls in the HI assays. Table S3. Results of the HI assay with H5N2 LPAIV. Table S4. Results of the HI assay with H5N3 LPAIV.
- Additionalfile308.01.21.pdf
Additional file 3: Sensitivity of H5 EB-ELISA relative to the commercial FluAC H5 test. Table S1. Detection of H5 subtype-specific antibodies in the reference antisera. Table S2. Detection of H5 subtype-specific antibodies in the experimental antisera.
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Reviewers invited
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This preprint is under consideration at Virology Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
A Novel Epitope-blocking ELISA for Specific and Sensitive Detection of Antibodies against H5-Subtype Influenza Virus Hemagglutinin
Violetta Sączyńska
Institute of Biotechnology and Antibiotics, Warsaw, Poland
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1828-6783
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 2
Posted 26 Jan, 2021
No community comments so far
Review #1 received
Received 24 Feb, 2021
Reviewer #1 agreed
On 13 Feb, 2021
Reviewers invited
Invitations sent on 14 Jan, 2021
Editor assigned
On 10 Jan, 2021
Submission checks complete
On 10 Jan, 2021
Editor invited
On 10 Jan, 2021
No comments provided
Editorial decision: Minor revision
On 20 Dec, 2020
Review #1 received
Received 20 Nov, 2020
Reviewer #1 agreed
On 11 Nov, 2020
Reviewers invited
Invitations sent on 10 Nov, 2020
Editor assigned
On 14 Oct, 2020
First submitted
On 13 Oct, 2020
Submission checks complete
On 13 Oct, 2020
Editor invited
On 13 Oct, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Virology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1