Name:

kd/koff

Category:

Binding

Property:

Rate Constant of Dissociation (koff)

Units:

1/s

Measurement Technique:

Surface Plasmon Resonance (SPR)

Buffers:

HBS-EP+ running buffer

Temperature:

None

pH:

None

Protein Concentration:

None

Wavelength:

None

Ionic Strength:

None

Details:

The Biacore T200 instrument (GE Healthcare) was used to assess the TGFβ binding affinity of the purified CAT192 WT and mutants. TGFβ1 and TGFβ2 were prepared and purified according to previously published procedures. TGFβ3 was purchased from R&D Systems. Each isoform was immobilized to a CM5 series S chip using the amine coupling kit (GE Healthcare). To determine the binding affinities, low density chips were prepared in order to minimize avidity effects from binding to the immobilized TGFβ homodimers. High-density chips were prepared to compare CAT192 WT binding. CAT192 (metelimumab) was tested in early experiments and the CAT192 IgG4 S228P construct was used for all other analysis. The S228P mutation was used to reduce Fab arm exchange. CAT192 WT and mutant Fabs or antibodies were always diluted in the HBS-EP+ running buffer (GE Healthcare). The surface was regenerated with 40 mM HCl as described previously. Binding sensorgrams were fit to a 1:1 binding model using the BiaEvaluation software (GE Healthcare) or Scrubber (BioLogic Software) to calculate binding affinities.