Production of H5-specific monoclonal antibodies and the development of a competitive enzyme-linked immunosorbent assay for detection of H5 antibodies in multiple species.
The hemagglutinin gene of an avian influenza virus (AIV) A/duck/NC/674964/07 (H5N2) was cloned and expressed in a baculovirus system (H5-Bac). In parallel, a recombinant hemagglutinin of A/Vietnam/1203/04 (H5N1) was expressed in mammalian cells, purified, and used for generation of H5-specific monoclonal antibodies (MAb). The purified H5-Bac was used to develop a competitive enzyme-linked immunosorbent assay (cELISA) to detect H5 antibodies in a species-independent approach using one of the established H5-specific MAbs as the competitor antibody. The cELISA performed with influenza antibody-free sera or with sera of animals infected with other than H5-encoding AIV showed no significant inhibition of H5-MAb binding, indicating high test specificity. In contrast, sera of poultry (chickens, turkeys, ducks) experimentally infected with H5-encoding AIV were able to significantly inhibit the binding of the MAb in a species-independent approach. Comparison of the results of the cELISA with results obtained by a hemagglutination inhibition assay showed a gradient of the sensitivity (turkeys > ducks > chicken). The described results show that H5-specific antibodies in sera can be detected in a species-independent approach by using a recombinant protein.
Department of Population Health, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA 30602, USA.
Daniel
D
Dlugolenski
Rüdiger
R
Hauck
Robert J
RJ
Hogan
Frank
F
Michel
Egbert
E
Mundt
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000662:veterinary*; D015967:Gene Expression Regulation, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology*; D006801:Humans; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology; D011200:Poultry / Q000097:blood*; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus