Influenza viruses (IFVs) recognize sialoglycans expressed on the host cell surface. To understand the mechanisms underlying tissue and host tropisms of IFV, it is essential to elucidate the molecular interaction of the virus with the host sialoglycan receptor. We established and applied a new monoclonal antibody, clone HYB4, which specifically recognizes the Neu5Ac alpha 2-3 determinant at the non-reducing terminal Gal residue of both glycoproteins and gangliosides to investigate the biochemical properties of IFV receptors in A549 cells. HYB4 significantly blocked virus binding to A549 cells in a dose-dependent manner. Virus overlay assay indicated that several glycoproteins with molecular masses of 80-120 kDa of A549 cells were commonly recognized by different subtypes of IFV, such as HI NI and H3N2. HI NI virus binding to the glycoproteins was diminished by pretreatment with either sialidase or PNGase F. On TLC-immunostaining experiments with HYB4, GM(3) ganglioside was only detected in A549 cells. Interestingly, this antibody bound to GM(3) gangliosides on TLC and plastic surfaces, but not on lipid bilayers. In comparison with the recognition of Maackia amurensis lectins, HYB4 exclusively recognized Neu5Ac alpha 2-3Gal beta 1-4GlcNAc residues expressed on glycoproteins. These results strongly suggest that N-linked sialoglycans with the Neu5Ac alpha 2-3 determinant on several glycoproteins are receptors for influenza virus in A549 cells. (C) 2013 Elsevier Inc. All rights reserved.