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Selection of Recombinant Antibodies 2472.3. Packaging of Phagemids1. 2X TY-AK: 2X TY, containing 100 µg/mL ampicillin, and 50 µg/mL kanamycin.2. Polyethylene glycol (PEG) solution: 20% (w/v) PEG 6000: Fluka (part of Sigma-AldrichChemie Gmbhl), München, Germany and 2.5 M NaCl.3. Phage dilution buffer: 10 mM Tris-HCl, 20 mM NaCl, and 2 mM ethylenediaminetetraaceticacid, pH 7.5.2.4. Titering1. 2X TY-GA agar plates: 2X TY-GA and 1.5% (w/v) agar–agar.2.5. ELISA of a Polyclonal Antibody Phage Suspension1. BSA: prepare a 10 mg/mL stock solution in PBS.2. Anti-M13, horseradish peroxidase (HRP)-conjugated monoclonal antibody(Amersham Bioscience; GE Healthcare, München, Germany).3. Tetramethylbenzidine (TMB) solution A: 10 g citric acid solved in 100 mL waterand add 9.73 g potassium citrate to 1 L water, pH 4.1.4. TMB solution B: 240 mg tetramethylbenzidine, 10 mL acetone, 90 mL ethanol,and 907 µL 30% H 2O 2.5. 1 N H 2SO 4.2.6. Production of Soluble Monoclonal Antibody Fragmentsin Microtiter Plates1. 96-well U-bottom polypropylene (PP) microtiter plates (Greiner, Germany).2. AeraSeal breathable sealing film (Excel Scientific: Wrightwood, CA).3. 2X TY-A containing 50 µM isopropyl-β-D-thiogalactopyranoside (IPTG).2.7. ELISA of Soluble Monoclonal Antibody Fragments1. Mouse α-His-tag monoclonal antibodies (α-Penta His, Qiagen, Germany).2. Mouse α-myc-tag monoclonal antibodies (9E10, Sigma, Germany).3. Mouse α-pIII monoclonal antibodies (PSKAN3, Mobitec, Germany).4. Goat α-mouse IgG serum (Fab specific) HRP-conjugated (Sigma, Germany).3. MethodsThe in vitro procedure for isolating antibody fragments by their bindingactivity was called “panning,” referring to the gold washers tool (26). The antigenis immobilized to a solid surface, such as nitrocellulose (e.g., ref. 27), magneticbeads (e.g., ref. 28), a column matrix (e.g., ref. 12) or, most widely used,plastic surfaces as polystyrole tubes (e.g., ref. 29) or 96-well microtiter plates(e.g., ref. 13). The antibody phage are incubated with the surface-bound antigen,followed by thorough washing to remove the vast excess of nonbinding antibodyphage. The bound antibody phage can subsequently be eluted and reamplified byinfection of E. coli. This amplification allows detection of a single molecular