Four different M13 phage libraries expressing 15-mer (X15), 30-mer (X30), 17-mer including a fixed cysteine residue (X8CX8), and 12-mer see more peptides including two fixed cysteine residues (XCX8CX) were employed [40]. Biopannings were performed as described previously [40] with some modifications. Briefly, 96-well microtiter plates (Becton Dickinson, Oxnard, CA) were incubated overnight at 4 °C with 100 μl LmmAbB2D4 at 5 μg/ml for the first two biopannings and 0.5 μg/ml for the last one, in 0.1 M NaHCO3, pH 8.6. The plates were then washed

with 0.05% Tween 20 in 50 mM Tris-buffered saline, pH 7.5 (TBS-T) and blocked with 3% BSA in TBS-T at 37 °C for 2 h. For the first biopanning, 1.5 × 1011 transducing units (TU) of phages expressing linear 15-mer (X15), 17-mer (X8CX8) and 30-mer (X30) peptides and 2.5 × 1010 TU of phages expressing constrained 12-mer (XCX8CX) peptides were incubated in TBS-T with

the immobilized LmmAbB2D4 overnight at 4 °C. Unbound phages were removed by washing with TBS-T. Bound phages were eluted with 0.1 M glycine, 1 mg/ml bovine serum albumin (pH 2.2) and neutralized with 2 M Tris–HCl, pH 9.0. Escherichia coli K91 cells were infected with the eluted phages and grown overnight at 37 °C. Phage particles were precipitated with 20% PEG 8000, 2.5 M NaCl overnight on ice, resuspended in 0.05 M Tris–HCl, 0.15 M NaCl, pH 7.5 and used for the next round of biopanning (2 × 1011 TU). After three rounds, phage clones were isolated JNJ-26481585 chemical structure and submitted to screening ELISA. Microtiter plate wells (Falcon) were coated with 100 μl sheep anti-M13 polyclonal antibody (10 μg/ml) in

0.1 M NaHCO3, pH 8.6, overnight at 4 °C. Plates were washed with 0.1% Tween 20-PBS (PBS-T) and blocked with 2% non-fat dry milk in PBS-T for 1 h at 37 °C. The wells were washed, and phages, diluted 1:1 in PBS-T, were incubated for 90 min at 37 °C. Phages able to bind LmmAbB2D4 at 10 μg/ml were detected by a horseradish peroxidise (HRP)-conjugated rabbit anti-mouse antibody (Sigma, St. Louis, MO, USA). The clones selected by LmmAbB2D4 Tacrolimus (FK506) were used in a new ELISA plate configuration to verify their reactivity with the monoclonal antibody. Plates were sensitized with 5 μg/ml LmmAbB2D4 in coating buffer, washed and blocked as described previously and incubated with phages at 1 × 1011 to 1 × 106 pfu/ml diluted in 0.05% Tween 20-PBS for 2 h at 37 °C. Binding was detected using a HRP-conjugated anti-M13 antibody (Sigma) diluted 1:3000 in blocking buffer. The single-stranded DNA was purified using the QIAprep Spin M13 protocol (Qiagen). Sequencing reactions were carried out according to the dideoxy chain termination method using the ABI Prism Kit using the ABI PRISM 377 (both from PE Applied Biosystems). The reverse primer 5′-TCGGCAAGCTCTTTTAGG-3′ was used for sequencing. The sequences obtained were translated and seventeen dodecapeptides corresponding to the XCX8CX library were identified.