H5 Antibody Detection by Blocking Enzyme-Linked Immunosorbent Assay Using a Monoclonal Antibody

Abstract

Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.

Galectin-1 Binds to Influenza Virus and Ameliorates Influenza Virus Pathogenesis

“…All human influenza viruses were propagated in MDCK cells (9). Influenza A/chicken/Taiwan/2838V/00 (H6N1) and influenza A/duck/Yunlin/04 (H5N2) viruses, which are two low-pathogenicity avian influenza viruses isolated from Taiwan (3,49), were propagated in embryonic chicken eggs and inactivated with 1% (vol/vol) of 0.1 M 2-bromoethylamine hydrobromide (BEI; Sigma, St. Louis, MO) dissolved in 0.2 N NaOH by constant shaking overnight at 37°C (1). Influenza A/WSN/33 (H1N1) virus was used in all of the experiments, unless stated otherwise.…”

Glycosylation at hemagglutinin Asn-167 protects the H6N1 avian influenza virus from tryptic cleavage at Arg-201 and maintains the viral infectivity

“…AIV strains A/chicken/Taiwan/2838V/00 (H6N1, abbreviated as 2838V) and A/duck/Yunlin/3233/04 (H5N2, abbreviated as 3233) are field isolates (Chen et al, 2008;Chou et al, 2011). These two subtypes are both low-pathogenic AIV strains (Wang and Wang, 2003).…”

Antigen-capture enzyme-linked immunosorbent assays for detection of different H5 avian Influenza A virus

“…The A5-2 to A5-7 strains were H5N2 isolated from chickens similar to the A5-1 strain. The E5-1 strain is a low pathogenic AIV that was isolated from healthy ducks during routine monitoring, 4 and has an HA sequence similar to highly pathogenic Eurasian lineage H5N1 AIV that occurred in Southeast Asian countries. Nine H6N1 AIV strains (H6-1 to -9) from chickens, and 1 Newcastle disease virus (TW-2/00 3 ) were also used in the current study to test the specificity of this assay.…”

“…The E5-1 H5N2 strain was selected for mAb production because it belongs to the Eurasian lineage as previously reported. 4 The percentage of the nucleic acid sequence , and anti-H5-6 (IgG2b), were previously produced in the authors' laboratory. 4 Stocked mAb-rich ascetic fluids were purified by 0%-40% ammonium sulfate precipitation and protein A affinity column.…”

“…Many commercial H5 vaccines (such as from the Intervet vaccine) and the H5N2 outbreaks that occurred in Taiwan in 2004 belong to the American H5 lineage. 4 The ability of the new ACE to differentiate between the American and Eurasian lineages of H5 AIV strains would be useful for detection when a new Eurasian H5 AIV intrudes into a country.…”