Increase in KD (M) value

Dear Team,

There is an increase in KD(M) value (refer data). What could be the possible reason for this change in KD/ increase in KD value over the time.
To overcome this problem, I have tried
1) Reducing the sample compartment temperature to 15 degree to check the impact of low temperature on stability of immobilized surface.
2) Mild condition regeneration buffer (10mM Glycine ph 3.0) 
3) Running buffer also used as a regeneration buffer but no significant result observed.


What could be the reason of this and how can I get the consistant KD (M) from 1st sample to last sample. Because, only first sample shows low KD while all other sample shows consistent KD around 4nM.
Kindly suggest.

I don't think the results are that different but the table suggest that you did four different experiments and four different fittings. Can you tell exactly what you did during the measurements.
Can you show the sensorgrams with the fitting of the results?

kind regards
Arnoud

Ok, so these are not four different experiments, not different fittings, not different samples.
It's a single experiment with one sample 4 times (4 determinants of same sample), as we do in a repeatability test (As per ICH Q2R2) and same fitting. 
In this experiment, first determinant is showing strong binding, and all other determinants of the same sample are showing slightly weak binding and consistent. 
My query was regarding the decrease in binding response i.e KD(M).
Why only first determinant is showing such binding every time and all other determinants of same sample in the same experiment is showing different and consistent binding? 

You say that one sample is injected 4 times in a row and each injection curve is fitted individually.
As recommended by the SPRpages always start with 1-3 dummy injections to 'prime' the instrument and ligand. This because the first injection/regeneration cycle is always different and should not be analysed.
Again (also for others who read this), please show sensorgrams, ka, kd and not only KD.

Kind regards
Arnoud

Dear Arnoud,
thank you for your continuous support.
Please go through the representative sensor grams of one of my experiments to justify the above results.
also, we give upto 5 primes before any experiment. And 6 startup cycles we have added in our method which runs before the actual injections of sample. But still, same trend of increase in KD(M) is observed.
Please refer data table and sensorgrams for better clarity of query.

In atteched picture
a) Group 1: Sample 1 which shows 5.559E-10.
b) Group 2: A representative sensorgram of other determianants of same sample shows 1.24e-09 and 1.35E-09
C) A data table with Ka, Kd, KD and Rmax is provided for better understanding.
Kindly check Rmax also for troubleshooting.
 

What do you with determinants"? Analyte?
Can you explain what you mean with "other determianants of same sample shows" ?

If Rmax changes significantly I suspect that the regeneration is not complete and leaving some analyte behind the first time. It can depend on the purity of the analyte.
Is there a reason to use this type of injection and not every analyte concentration separately in a cycle?

regards
Arnoud