(Beijing, China), as well as a mouse monoclonal antibody against H5N1. mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 SRT 1720 Hydrochloride but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein Ywhaz by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus. Introduction Influenza is a highly contagious disease caused by viruses that belong to the family gene of HPAI H5N1 virus belongs to the A/goose/Guangdong/1/96 (H5N1) lineage, and all HPAI H5N1 viruses have a characteristic multibasic sequence in the HA CS [15]. Although there is no evidence that HPAI H5N1 viruses transmit between mammals, an experimentally mutated HPAI H5N1 virus has been transmitted via droplets in a ferret model [16]. Thus, the scientific and public health communities need to prepare for a potential HPAI H5N1 pandemic. Hence, the diagnosis and subtyping of HPAI SRT 1720 Hydrochloride H5N1 viruses are high priorities for public health. For detecting HPAI H5N1 virus and diagnosing influenza, a number of specific monoclonal antibodies have been developed [17], [18]. However, because the primary structure of H5N1 HA is highly homologous to H1 subtype viruses, these monoclonal antibodies might have considerable cross-reactivity [19]. In the present study, we report several unique recombinant Fab fragments obtained from an immunized phage display library that target the CS peptide of HA derived from HPAI H5N1 virus (HA331), and we discuss their potential applications in diagnostics. Results Selection of recombinant anti-HA331 Fab fragments by phage library screening The strategy for making anti-HA331 monoclonal antibodies is shown in Fig. 1, A and B. First, mice were SRT 1720 Hydrochloride immunized with the HA331-bovine serum albumin (BSA) conjugate. After the quantitation of peptide-specific antibodies in sera, the variable region genes of the antibody heavy (VH) and light (VL) chains were prepared and cloned to a phagemid vector to perform phage display selection. We used a pDong1/Fab phagemid vector that was previously used to clone anti-T4 Fab fragments [20]. Using this system, the cDNA fragments for VH and VL were iteratively cloned into pDong1/Fab, and a bacterial library with a diversity of 5106 was used to make the Fab-phage library. After three rounds of biopanning selection, an ELISA with immobilized HA331 peptide was performed with the original (R0) and selected (R1CR3) libraries to confirm the enrichment of HA331-specific phages. The signals for R0, R1, R2 and R3 phages increased gradually in the ELISA, confirming the enrichment of specific Fab-phages (data not shown). Open in a separate window Figure 1 Selection of anti-HPAI HA antibodies.(A) The structure of BSA-MBS-HA331 used for mice immunization. (B) Flow chart for the development of monoclonal Fabs. The RNA extracted from the spleen cells of immunized mice was used for RT-PCR, which produced VH and VL cDNAs. These fragments were used to make the pDong1 phagemid library that was subsequently used for biopanning the phage libraries. (C) ELISA of the binding of positive Fab-phages to HA 331 peptide and HA proteins. HA331: cleavage site peptide; MBS: m-maleimidobenzoic acid N-hydroxysuccinimide ester; BSA: bovine serum albumin; H1N1-HA: recombinant A/California/04/2009 H1N1 HA; H5N1-HA: recombinant A/Vietnam/1194/2004 H5N1 HA. Monoclonal antibody selection The phages obtained at round 3 were used to infect bacteria, and ninety-six clones were selected and cultivated for making Fab-phage. When an ELISA was performed, four clonesA3, A4, D4, and D8showed strong signal against immobilized streptavidin (SAv)-HA331, and these were further analyzed. When the.