scholarly journals Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses

2012 ◽  
Vol 45 (3) ◽  
pp. 243-247 ◽  
Author(s):  
Yi-Tung Chen ◽  
Zak Tsao ◽  
Shu-Ting Chang ◽  
Ron-Huay Juang ◽  
Lih-Chiann Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Avian Influenza ◽  
Immunosorbent Assay ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Avian Influenza Viruses ◽  
Antigen Capture

scholarly journals Rapid Detection of H5N1 Subtype Influenza Viruses by Antigen Capture Enzyme-Linked Immunosorbent Assay Using H5- and N1-Specific Monoclonal Antibodies

2009 ◽  
Vol 16 (5) ◽  
pp. 726-732 ◽  
Author(s):  
Hui-Ting Ho ◽  
Hong-Liang Qian ◽  
Fang He ◽  
Tao Meng ◽  
Milene Szyporta ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Rapid Detection ◽  
Conformational Epitope ◽  
Cross Reactivity ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Influenza B ◽  
H5n1 Subtype ◽  
Antigen Capture

ABSTRACT Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype has caused devastating damage to poultry flocks and sporadic human H5N1 infections. There is concern that this virus subtype may gain transmissibility and become pandemic. Rapid diagnosis and surveillance for H5N1 subtype viruses are critical for the control of H5N1 infection. In this study, we report a robust antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) based on H5- and N1-specific monoclonal antibodies (MAbs) for the rapid detection of H5N1 subtype viruses. The H5 hemagglutinin (HA)-specific MAb (2D9) targets a conformational epitope which recognized multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8. The N1 neuraminidase (NA)-specific MAb (8H12) recognized a linear epitope comprising the sequence AELPF. This epitope was 99% conserved in the NA of 708 analyzed H5N1 viruses, while the epitope was absent in NAs of subtypes N2 through N9. The specificity of the AC-ELISA was examined by using 41 H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. The estimated detection limit was 1 to 2 HA titers. It is concluded that this H5N1 AC-ELISA can simultaneously detect H5 and N1 subtype antigens, eliminating the need for secondary testing for the NA subtype. Implementation of this assay in ELISA-like formats suitable for field use, such as dot ELISA, immunofiltration, or electrochemical biosensor technologies, would provide dual on-site detection of H5 and N1 in clinical or environmental specimens.

scholarly journals Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody

2007 ◽  
Vol 14 (5) ◽  
pp. 617-623 ◽  
Author(s):  
Qigai He ◽  
Sumathy Velumani ◽  
Qingyun Du ◽  
Chee Wee Lim ◽  
Fook Kheong Ng ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Immunosorbent Assay ◽  
Influenza Viruses ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Attractive Alternative ◽  
H5n1 Avian Influenza Virus ◽  
Antigen Capture

ABSTRACT The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1) expressed in bacteria or immunized with concentrated H5N2 virus yielded a panel of hybridomas secreting MAbs specific for influenza virus HA. The reactivity of each MAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, which secrete immunoglobulin G (IgG) and IgM, respectively, were used as the capture antibodies and pooled hyperimmune guinea pig serum IgG served as the detector antibody. The specificity of the optimized AC-ELISA was evaluated by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens containing AIV subtype H5 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limits of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1 protein and 124, 62, and 31 50% tissue culture infective doses of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in the AC-ELISA, and the results were confirmed by reverse transcription-PCR. The tracheal swab samples from H9N2-infected chickens did not give positive signals. Taken together, the newly developed MAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H5 AIV.

scholarly journals Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yixin Xiao ◽  
Fan Yang ◽  
Fumin Liu ◽  
Hangping Yao ◽  
Nanping Wu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Rapid Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunochromatographic Test ◽  
H9n2 Subtype ◽  
Antigen Capture

Abstract Background The H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV. Methods In this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV. Results The AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 μl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 μl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively. Conclusions The developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.

scholarly journals Production of highly and broad-range specific monoclonal antibodies against hemagglutinin of H5-subtype avian influenza viruses and their differentiation by mass spectrometry

2018 ◽  
Vol 15 (1) ◽  
Author(s):  
Violetta Sączyńska ◽  
Anna Bierczyńska-Krzysik ◽  
Violetta Cecuda-Adamczewska ◽  
Piotr Baran ◽  
Anna Porębska ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibodies ◽  
Avian Influenza ◽  
Influenza Viruses ◽  
Avian Influenza Viruses ◽  
H5 Subtype

scholarly journals Inactivated H7 Influenza Virus Vaccines Protect Mice despite Inducing Only Low Levels of Neutralizing Antibodies

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Ram P. Kamal ◽  
Kristy Blanchfield ◽  
Jessica A. Belser ◽  
Nedzad Music ◽  
Wen-Pin Tzeng ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Hemagglutination Inhibition ◽  
Neutralizing Antibodies ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Antibodies ◽  
Avian Influenza Viruses ◽  
Homologous Virus ◽  
Antibody Titers ◽  
Low Levels

ABSTRACT Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutination inhibition (HI) and microneutralization (MN) assays, H7 viruses and vaccines are found to induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate prepandemic vaccines. We have previously shown that purified recombinant H7 HA appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. In this study, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and neuraminidase (NA) from 3 different H7 viruses [A/Shanghai/2/2013(H7N9), A/Netherlands/219/2003(H7N7), and A/New York/107/2003(H7N2)] or with human A(H1N1)pdm09 (A/California/07/2009-like) or A(H3N2) (A/Perth16/2009) viruses. Mice produced equivalent titers of antibodies to all viruses as measured by enzyme-linked immunosorbent assay (ELISA). However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for prepandemic vaccines. IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of prepandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low titers in HI and MN assays, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus vaccines (WIVs) were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was nonneutralizing in vitro. Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

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