Situation: Very slow off rate binder with koff <1e-4 or -5. KD in low nM to fM range around LOD of S200. Testing using single cycle kinetics, low target density, Rmax about 10-15 RU using reversible biotin capture CAPture kit, at 25 degree (with top Analyte conc ~ 1uM) and then at 37 degree ( with top analyte conc ~5 nM, longer contact time and dissociation time). I got two sets of kinetic data with clear dissociation at 37 degree vs almost no dissociation at 25 degree. Should the data at 37 degree be only used as validation for data at 25 degree or should 37 degree data be the primary data for future SAR ?
37 degree: ka 4 X e6, kd 3 X e-3, KD 0.8 nM, Rmax 16 RU, Chi2 0.06
25 degree: ka 9 X e5, kd 3 X e-4, KD 0.4 nM, Rmax 10 RU, Chi2 0.07