k_off

Binding

Rate Constant of Dissociation (koff)

1/s

Surface Plasmon Resonance (SPR)

HBS-EP buffer (10 mM HEPES ( pH 7.4), 150 mM NaCl, 3.4 mM EDTA, 0.05% surfactant P20)

25 C

None

None

None

None

Binding analysis using SPR
SPR experiments were performed by using a BIAcoreT100 (GE Healthcare). Troponin I from human cardiac muscle (Funakoshi, Tokyo, Japan) was immobilized on research-grade CM4 chips (GE Healthcare). Each purified scFv protein was buffer-substituted with HBS-EP buffer (10 mM HEPES ( pH 7.4), 150 mM NaCl, 3.4 mM EDTA, 0.05% surfactant P20) and injected over the immobilized troponin I at a flow rate of 50 ml/ min. The data for experimental responses were normalized by subtracting the response for a blank cell in which bovine serum albumin alone was immobilized. BIA evaluation software version 2.0.2 (GE Healthcare) was used to analyze the data. Kinetic parameters were calculated by means of a global fitting analysis with the assumptions of the 1 : 1 Langmuir binding model.