koff

Binding

Rate Constant of Dissociation (koff)

1/s

Surface Plasmon Resonance (SPR)

HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) P20)

None

7.4

10–0.5 nM; 5 µM–50 nM

None

None

Before surface plasmon resonance (SPR) binding experiments, IMAC-purified VHHs were subjected to de-salting and further purification by size-exclusion chromatography (SEC). Approximately 500 µg of each VHH was injected over a Superdex 75 Increase 10/300 GL SEC column (GE Healthcare) in Biacore running buffer HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) P20; GE Healthcare) under the control of an ÄKTA FPLC at 0.8 mL/min. Monomeric VHH fractions were collected and analyzed for binding to TcdA using a Biacore 3000 SPR instrument (GE Healthcare). Approximately 4,500 resonance units (RUs) of TcdA (List Biological Laboratories, Campbell, CA) were immobilized on a CM5 sensor chip (GE Healthcare) using the conditions previously described36. HBS-EP running buffer was used for all binding studies and regeneration of TcdA surfaces. Next, various dilution ranges of VHHs (as low as 10–0.5 nM to as high as 5 µM–50 nM) were injected over immobilized TcdA at a flow rate of 40 µL/min with 120 s contact time and 600 s dissociation time. Reference-subtracted sensorgrams were analyzed with BIAevaluation software (GE Healthcare) and fit to a 1:1 binding model. In cases where rapid kon and koff rate constants were observed, equilibrium dissociation constants (KDs) were determined by steady-state analysis. All mutant A26.8 VHHs were run in duplicates and the parent A26.8 VHH in quintuplicate.