High concentration curve

Hello. I ran this experiment on a CM5 chip with an anti-mouse surface. mAB had an RL of ~480.. Mw analyte - 8400 Da. Contact - 180 sec, Diss. - 540 sec. Prepared the following conc. : 20, 4, 0.8, 0.16, 0.03 nM. Noticed that the 20nM did (Figure 1) not show much curvature, too high, so I deleted this top point (Figure 2), but I am getting QC errors that the ka and kd approaching limits. Figure 3 is data summary. I am thinking of re-running the assay by deleting the 20nM and preparing 4, 2, 1, 0.5, 0.25nM. But any additional suggestions would be most helpful. Thanks.

Hi,
Since the high association and slow dissociation the QC errors I can understand.
First a longer dissociation time can make the values more reliable. Remember that 5% deacy of the initial valu at start of dissociation is recomended. You can try the short & long system ( www.sprpages.nl/experiments/strategy ). Like wise the association is very strong. The 4 nM is >5000x KD which indicates that fitting can be difficult. If possible try 10x - 0.1x KD based on the last fitting and if possible extend the association time. Due to the low dissociation rate it takes long to get some curvature, ecspecially for the lower concentrations.
Be prepared that this interaction is outside the limits of the system.

Arnoud

Hi Arnoud, So I did a follow up experiment in which I injected 2 different concentrations of mAB, with FC2 (Figure 1A) being lower than FC3 (Figure 2A). I was concerned that I maybe immobilizing too much on the surface. I also extended the contact to 360sec and extended the dissoc. to 30min. I also adjusted my conc. range to the following conc's: 0.125, 0.25, 0.5, 1.0, 2.0nM. I am now getting some curvature on the ka and what it looks like some decay on kd? The signal is pretty choppy I suppose due to the low signal. Is this expected? Curve fitting still looks decent. Using T200. I am still getting KD's in the E-12M range. Is this the best I can do? I've done so many scouting runs already.. Thanks.

Hi,
This look pretty well.
The highest analyte concentration has a nice curvature which will constrain the ka. The other curves show that the binding is following the analyte concentrations. The dissociation looks ok. And two different ligand concentrations give the same results.
I would say: well done!

Arnoud

Thanks Arnoud for your help!

Hi Arnoud,
So I repeated the experiment using the same mAB, same anlayte conc., and I am seeing a difference in KD. Previously getting E-12 M, now getting E-13M. Off-rate looks different between the two. Also this result is about 0.13pM. Is this realistic for mAB's? Kindly comment. Thanks.