Characterization and identification of a novel single-domain antibody targeting cyclophilin A. (A) Expression and purification of anti-CypA single-domain antibody (sdAb) A1. The sdAbA1 antibody was purified from periplasmic lysates by Ni-NTA columns followed by gel filtration. The protein sample collected during purification was size fractionated by SDS-PAGE and stained with Coomassie blue. M, molecular weight markers, size indicated in kDa; lane 1, total proteins in periplasmic lysates as loaded onto the column; lane 2, flowthrough obtained from the Ni-NTA column; lanes 3 and 4, proteins eluted from the Ni-NTA column and Superdex 75 prep grade column respectively. (B) Binding of sdAbA1 to cyclophilin A (CypA) was detected by enzyme-linked immunosorbent assay. Purified CypA (1 μg/ml) was coated on 96-well plates and incubated with different concentrations of sdAbA1 or sdAbE2 (control sdAb). Binding of sdAbs was detected with horseradish peroxidase-conjugated anti-his antibody, visualized with TMB substrate and the plate was read at 450 nm. (C) Binding kinetics assay of sdAbA1 by surface plasmon resonance. (D) sdAbA1 inhibits the peptidyl-prolyl cis–trans isomerase (PPIase) activity of CypA using a chymotrypsin-coupled assay. CypA (0.1 μM) is shown as a positive control for PPIase activity and the absence of CypA and addition of CsA are used as blank control and negative control respectively. The presence of sdAbA1 inhibits the rate of cleavage of the tetrapeptide substrate in a dose-dependent manner compared with CypA alone. (A) to (D) Data are representative results from three independent experiments. ka, association rate constant; kd, dissociation rate constant; kD the equilibrium dissociation constant .