Kinetics vs. Affinity Analysis

I use the Biacore T200 with the CM5 chip. Recently, I ran an experiment where I saw an extremely high off-rate and on-rate. In fact, the Biacore Evaluation software gave the message that binding constants cannot be uniquely determined (I'm assuming because of the fast kinetics?). I went ahead and did affinity analysis (you can see both below) and found the KD to be 2.61 uM with X^2 of 1.85 and Rmax of 44. My immobilization level was a bit low but I decided to go ahead because my analyte is fairly large (~15 kDa) and I didn't need super high immobilization. I did pre-concentration but could always repeat to verify it was correct. I also have my ligand in citrate buffer (~pH 5) which I realize is a bit different than typical as usually sodium acetate buffer is used.

Do you think this is trustworthy data? If publishing the affinity data but not kinetics, how would I justify? Just wondering how normal it is to see fast on/off rate but still have specific interaction.

Hi,
When I look at the affinity analysis it looks good. The curve goes well toward Rmax and the K_D is well within the measured range.
You have a slight negative drift in the sensorgram but that should not of great concern but consider double referencing if you did this not already.
One thing you can do is to create a second channel with a higher ligand concentration and flow the analyte over both channels. Both affinity curves should give you the same number for the K_D but with different Rmax.
Is there another way to confirm your interaction? Something functional?
The citrate pH 5.0 should not be more detrimental than the acetate buffer to the activity of the ligand.

Typical pharmaceutical compounds have this kind of profile so this is what it is.

Also look at: www.sprpages.nl/troubleshooting/low-affinity

Kind regards
Arnoud

I agree that the affinity plot and the sensorgrams look good. I routinely see this type of profile with drug small molecules (i.e. warfarin) binding to albumin. In these cases, I also use the affinity plot to interpolate KD. Your chi2 is acceptable and I'm not sure what your immobilization level is, but if an Rmax of 44 makes sense, and your KD is within the concentration range that you analyzed, then I think it is a solid experiment. Definitely worth repeating though for publication and any claims you want to publish. You could also try capturing your ligand in a different way (biotinylated ligand to avidin chip, his-tag ligand to Ni-NTA chip, etc. if you are worried about immobilization level). Also, in my experience, as long as you have enough ligand immobilized to generate a concentration-dependent response, then lower immobilization levels are better for kinetics experiments.