Development of an antigen-capture enzyme-linked immunosorbent assay and immunochromatographic strip based on monoclonal antibodies for detection of H6 avian influenza viruses

2020 ◽  
Vol 165 (5) ◽  
pp. 1129-1139
Author(s):  
Fan Yang ◽  
Yixin Xiao ◽  
Lihua Xu ◽  
Fumin Liu ◽  
Hangping Yao ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Avian Influenza ◽  
Immunosorbent Assay ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Avian Influenza Viruses ◽  
Immunochromatographic Strip ◽  
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 Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ge Li ◽  
Xun Wang ◽  
Qingmei Li ◽  
Jifei Yang ◽  
Xiao Liu ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Point Of Care ◽  
Influenza Viruses ◽  
Control Line ◽  
Avian Influenza Viruses ◽  
Igg Antibody ◽  
Immunochromatographic Strip ◽  
Live Poultry ◽  
Pathogenic Viruses ◽  
H7 Subtype

Abstract Background H7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection. Methods The H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared. Results The analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples. Conclusion The McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.

scholarly journals Development of an Immunochromatographic Strip for Rapid Detection of H9 Subtype Avian Influenza Viruses

2008 ◽  
Vol 15 (3) ◽  
pp. 569-574 ◽  
Author(s):  
Fuhu Peng ◽  
Zheng Wang ◽  
Shuhui Zhang ◽  
Renwei Wu ◽  
Sishun Hu ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Rapid Detection ◽  
Clinical Symptoms ◽  
High Specificity ◽  
Cloacal Swab ◽  
Influenza Viruses ◽  
Control Line ◽  
Avian Influenza Viruses ◽  
Immunochromatographic Strip

ABSTRACT An immunochromatographic strip was developed for the detection of the H9 subtype of avian influenza viruses (H9AIVs) in poultry, using two monoclonal antibodies (MAb), 4C4 for H9AIV hemagglutinin (HA) and 4D4 for nucleoprotein. The 4C4 MAb was labeled with colloidal gold as the detection reagent, and the 4D4 MAb was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane. In comparison with the HA and HA inhibition (HI) tests, the strip was specific for the detection of H9AIV, with a sensitivity at 0.25 HA units within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Evaluation of the strip with experimental tracheal and cloacal swab samples collected from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 postinoculation, earlier than the appearance of clinical symptoms. Application of the strip for the analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms had chickens that were infected with H9AIV. Further characterization of 10 positive and 30 negative randomly selected samples showed that no single sample was false positive or negative, as determined by the standard virus isolation and HI assays. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity, and stability. This finding, together with the advantages of rapid detection and easy operation and without the requirement for special skills and equipment, makes the strip suitable for onsite detection and the differentiation of H9AIVs from other viruses in poultry.

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.

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