title: Multimeric recombinant M2e protein-based ELISA: a significant improvement in differentiating avian influenza infected chickens from vaccinated ones.
abstract: Killed avian influenza virus (AIV) vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA) in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e) of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e) for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006) was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.
authors.affiliation: School of Animal and Veterinary Sciences, The University of Adelaide, South Australia, Adelaide, Australia.
authors.forename: Farshid
authors.initials: F
authors.lastname: Hadifar
authors.affiliation: School of Veterinary Science, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Jagoda
authors.initials: J
authors.lastname: Ignjatovic
authors.affiliation: Indonesian Research Centre for Veterinary Science, Bogor, West Java, Indonesia.
authors.forename: Simson
authors.initials: S
authors.lastname: Tarigan
authors.affiliation: Indonesian Research Centre for Veterinary Science, Bogor, West Java, Indonesia.
authors.forename: Risa
authors.initials: R
authors.lastname: Indriani
authors.affiliation: School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Esmaeil
authors.initials: E
authors.lastname: Ebrahimie
authors.affiliation: School of Animal and Veterinary Sciences, The University of Adelaide, South Australia, Adelaide, Australia.
authors.forename: Noor Haliza
authors.initials: NH
authors.lastname: Hasan
authors.affiliation: School of Animal and Veterinary Sciences, The University of Adelaide, South Australia, Adelaide, Australia.
authors.forename: Andrea
authors.initials: A
authors.lastname: McWhorter
authors.affiliation: School of Animal and Veterinary Sciences, The University of Adelaide, South Australia, Adelaide, Australia.
authors.forename: Sophie
authors.initials: S
authors.lastname: Putland
authors.affiliation: Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
authors.forename: Abdulghaffar
authors.initials: A
authors.lastname: Ownagh
authors.affiliation: School of Animal and Veterinary Sciences, The University of Adelaide, South Australia, Adelaide, Australia.
authors.forename: Farhid
authors.initials: F
authors.lastname: Hemmatzadeh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D015153:Blotting, Western; D002645:Chickens / Q000276:immunology / Q000821:virology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology / Q000502:physiology*; D007252:Influenza Vaccines / Q000276:immunology*; D005585:Influenza in Birds / Q000097:blood / Q000517:prevention & control / Q000821:virology*; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000086082:Viroporin Proteins
chemical_list: D000956:Antigens, Viral; D007252:Influenza Vaccines; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins