title: Development and evaluation of an M2-293FT cell-based flow cytometric assay for quantification of antibody response to native form of matrix protein 2 of influenza A viruses.
abstract: Matrix protein 2 (M2) of influenza A viruses is an attractive target for the development of broadly cross-protective influenza vaccines and therapeutic antibodies. The available evidence suggests that antibodies reactive to the natural tetrameric form of M2 proteins, rather than those to synthetic peptides of M2 ectodomain (M2e), best correlate with M2-mediated immune protection. However, the current ability to quantify strain-specific and/or subtype-cross-reactive M2 antibodies against the natural form of M2 antigens from influenza A viruses of different host origin is limited. In the present study, we generated a panel of 293FT transfected cell lines stably expressing full-length tetrameric forms of M2 molecules from human, avian and the swine-origin 2009 pandemic H1N1 influenza A virus, respectively, and developed an M2-293FT cell line-based flow cytometric assay (M2-FCA). Side-by-side comparison of M2-FCA with a synthetic M2e peptide-based indirect ELISA (M2e-ELISA) reveals that M2-FCA is highly efficient in quantifying both M2e sequence-specific and cross-reactive antibodies to the native form of M2 antigens. In contrast, promiscuity was evident when specificity and cross-reactivity of anti-M2 antibodies were assessed by M2e-ELISA. These results demonstrate that M2-FCA represents a rapid, simple and sensitive method to quantitatively assess specificity and cross-reactivity of anti-M2 antibodies after infection or vaccination.
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA. wzhong@cdc.gov
authors.forename: Weimin
authors.initials: W
authors.lastname: Zhong
authors.forename: Ju
authors.initials: J
authors.lastname: He
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Tang
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Xiuhua
authors.initials: X
authors.lastname: Lu
authors.forename: Hui
authors.initials: H
authors.lastname: Zeng
authors.forename: Abbas
authors.initials: A
authors.lastname: Vafai
authors.forename: Tong-Ming
authors.initials: TM
authors.lastname: Fu
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Katz
authors.forename: Kathy
authors.initials: K
authors.lastname: Hancock
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000032:analysis* / Q000276:immunology; D000918:Antibody Specificity; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000276:immunology*; D008969:Molecular Sequence Data; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000276:immunology*; D000086082:Viroporin Proteins
chemical_list: D000914:Antibodies, Viral; D000939:Epitopes; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins