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Determination of dissociation constant (KD) via isothermal titration calorimetry (ITC). Purified monobodies were injected into the sample cells loaded with their respective targets. Binding events elicited a heat change, which was then graphed as a function of molar ratio. GE Healthcare software (GE Healthcare; Piscataway, NJ, USA) was used to extrapolate the KD value. Both clones exhibited affinities in the single-digit nanomolar range.
