So I have this receptor and antibody interaction that I am studying but I see that in the method I have developed the results are randomly inconsistent- what i mean is when I run a set of three runs one run randomly shows a result which is shows a red cross for QC parameters (BTW I use Biacore T200/T100). I feel I am missing something very minor that is giving me this problem but it has been a nightmare to submit my work!
So the method is AntiHis capture from GE, I capture ~50-80 RU of the receptor which is quite stable (thank god!) and the antibody range is fixed. I am using HBS-EP buffer (ready to use ones) which is pH adjusted to 6.0 (because of the receptor property, it does not bind to antibody at a pH of 7.0) the method is run at 15 C (set analysis temp.)- beacuse i want to retard the dissociation rate otherwise its too fast at 25C to be captured by Biacore!
I will attach the images of two results that are from the same run one passes and the other fails for QC tabs as well as the kinetic values are absurd!
Any help would be appreciated (as usual, u have helped me a lot for my thesis- teh blog, this forum)
In the second fitting the algorithm seems to get stuck and reports these wrong values. In the fitting parameters you can adjust the initial values of the ka and kd (10 times higher or lower) and try the fit again to see if the values persist.
Definitely the second fitting is wrong.
Each method has a lower and higher boundary that defines the range of values that can be measured. I don't know by heart but i think that for the ka the upper limit is around 10^6 M-1s-1.
Since your curves seems to reach steady state very well, I would also check the if the steady state affinity corresponds with the fitted value of the kinetic injections.