Ant, and the mixture was incubated on ice for 1 h. After incubation, the mixture was centrifuged at 6,0006 g for 30 min. The pellet was resuspended in 2 ml of PBS and centrifuged at 15,0006 g for 10 min to pellet cell debris. The supernatant, containing Fabdisplaying phage, was collected.Immunization of mice with BSA-HA331 conjugateThe peptide for the H5N1 hemagglutinin CS, HA331 (NH2CTGLRNSPQRERRRRKKR-COOH), and its N-terminal biotinylated derivative bioHA331 (Bio-NH2TGLRNSPQRERRRRKKR-COOH) were synthesized by Genscript (Piscataway, NJ). For the coupling of HA331 peptide to BSA, five hundred microliters of BSA (10 mg/ml in 10 mM phosphate buffer, pH 7.4) was mixed with 70 ml of m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (3 mg in 200 ml of CI 1011 web dimethylformamide) at 25uC with stirring, followed by gel filtration through a PD-10 column (GE Healthcare) equilibrated with 50 mM sodium phosphate, pH 6.0. One milliliter of filtered BSA/MBS was added to 100 ml of HA331 solution (4 mg/ml in H2O), incubated for 3 h at room temperature, and purified with a PD-10 column equilibrated with phosphate-buffered saline (PBS) (KH2PO4, 1.47 mM; 61177-45-5 price Na2HPO4, 8.10 
mM; NaCl, 136.89 mM; KCl, 2.68 mM). Mice were immunized with the BSA-HA331 conjugate by Scrum Co. Ltd. (Tokyo, Japan). In brief, two inbred BALB/c mice were immunized four times at 2-week intervals with 150 ml of 500 mg/ml BSA-HA331 in Freund’s complete adjuvant. After the last immunization, blood samples were used to check for the presence of HA331-specific antibodies by an enzyme linked immunosorbent assay (ELISA). This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the School of Engineering, the University of Tokyo (Permit No. K18-1). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Phage ELISAThe microplate (Nunc, Langenselbold, Germany) was coated overnight with 100 ml per well of streptavidin (SAv, 10 mg/ml), BSA (10 mg/ml; Sigma), recombinant influenza A virus HA (0.5 mg/ml), and H5N1 HA fused with Fc (A/Vietnam/1203/ 2004; 0.5 mg/ml) in PBS at 4uC overnight. After washing, 1 mg/ml bio-HA331 in PBS was added and incubated for 30 min for the SAv-immobilized wells. Each well was blocked at 25uC for 2 h with 2 skimmed milk in PBS (MPBS), washed three 15755315 times with 0.1 Tween 20 in PBS (PBST), and incubated with 100 ml/well of MPBS containing 109?010 colony forming units (cfu) of Fabdisplaying phages at 25uC for 1 h. The plate was washed three times with PBST and incubated with 100 ml/well of HRPconjugated anti-M13 monoclonal antibody (GE Healthcare) diluted 5000-fold in MPBS at 25uC for 1 h. After three PBST washes, the signal was developed 
with TMBZ solution [100 mg/ml 3,39,5,59-tetramethylbenzidine (Sigma) and 0.04 ml/ml H2O2 in 100 mM NaOAc, pH 6.0], and the reaction was stopped with 50 ml/well of 10 sulfuric acid. The absorbance was read using a Model 680 microplate reader (Bio-Rad, Tokyo, Japan) at 450 nm with 655 nm as a control.Selection of HA331 pecific antibody-phage from phage libraryAntibody selection from the phage display library was performed on a microplate on which SAv-HA331 was immobilized. After blocking the microplate with MPBS for 2 h, 100 ml of phage solution (1012 cfu/ml in PBS) was added and incubated for 1 h at.Ant, and the mixture was incubated on ice for 1 h. After incubation, the mixture was centrifuged at 6,0006 g for 30 min. The pellet was resuspended in 2 ml of PBS and centrifuged at 15,0006 g for 10 min to pellet cell debris. The supernatant, containing Fabdisplaying phage, was collected.Immunization of mice with BSA-HA331 conjugateThe peptide for the H5N1 hemagglutinin CS, HA331 (NH2CTGLRNSPQRERRRRKKR-COOH), and its N-terminal biotinylated derivative bioHA331 (Bio-NH2TGLRNSPQRERRRRKKR-COOH) were synthesized by Genscript (Piscataway, NJ). For the coupling of HA331 peptide to BSA, five hundred microliters of BSA (10 mg/ml in 10 mM phosphate buffer, pH 7.4) was mixed with 70 ml of m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (3 mg in 200 ml of dimethylformamide) at 25uC with stirring, followed by gel filtration through a PD-10 column (GE Healthcare) equilibrated with 50 mM sodium phosphate, pH 6.0. One milliliter of filtered BSA/MBS was added to 100 ml of HA331 solution (4 mg/ml in H2O), incubated for 3 h at room temperature, and purified with a PD-10 column equilibrated with phosphate-buffered saline (PBS) (KH2PO4, 1.47 mM; Na2HPO4, 8.10 mM; NaCl, 136.89 mM; KCl, 2.68 mM). Mice were immunized with the BSA-HA331 conjugate by Scrum Co. Ltd. (Tokyo, Japan). In brief, two inbred BALB/c mice were immunized four times at 2-week intervals with 150 ml of 500 mg/ml BSA-HA331 in Freund’s complete adjuvant. After the last immunization, blood samples were used to check for the presence of HA331-specific antibodies by an enzyme linked immunosorbent assay (ELISA). This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the School of Engineering, the University of Tokyo (Permit No. K18-1). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Phage ELISAThe microplate (Nunc, Langenselbold, Germany) was coated overnight with 100 ml per well of streptavidin (SAv, 10 mg/ml), BSA (10 mg/ml; Sigma), recombinant influenza A virus HA (0.5 mg/ml), and H5N1 HA fused with Fc (A/Vietnam/1203/ 2004; 0.5 mg/ml) in PBS at 4uC overnight. After washing, 1 mg/ml bio-HA331 in PBS was added and incubated for 30 min for the SAv-immobilized wells. Each well was blocked at 25uC for 2 h with 2 skimmed milk in PBS (MPBS), washed three 15755315 times with 0.1 Tween 20 in PBS (PBST), and incubated with 100 ml/well of MPBS containing 109?010 colony forming units (cfu) of Fabdisplaying phages at 25uC for 1 h. The plate was washed three times with PBST and incubated with 100 ml/well of HRPconjugated anti-M13 monoclonal antibody (GE Healthcare) diluted 5000-fold in MPBS at 25uC for 1 h. After three PBST washes, the signal was developed with TMBZ solution [100 mg/ml 3,39,5,59-tetramethylbenzidine (Sigma) and 0.04 ml/ml H2O2 in 100 mM NaOAc, pH 6.0], and the reaction was stopped with 50 ml/well of 10 sulfuric acid. The absorbance was read using a Model 680 microplate reader (Bio-Rad, Tokyo, Japan) at 450 nm with 655 nm as a control.Selection of HA331 pecific antibody-phage from phage libraryAntibody selection from the phage display library was performed on a microplate on which SAv-HA331 was immobilized. After blocking the microplate with MPBS for 2 h, 100 ml of phage solution (1012 cfu/ml in PBS) was added and incubated for 1 h at.
