When capturing a ligand with an antibody you can have two reactions. Look at
for an explanation.
In addition, in your second setup the ligand is first inhibited before immobilization. In the first setup you uses un-inhibited analyte. There is a possibility this will give rise to affinity differences.
A 5-fold difference in KD is something but I wonder if it is significant. How hard is this value?
One remark: When immobilization decreases mobility is will decrease affinity -> there are less binding opportunities so you need more analyte so the KD will be higher.
I am sorry, I wanted to say that with immobilization strategy 2 the affinity may be affected so the KD will increase.
I agree with you that a 5 fold increase in KD should not be significant, this may be relative. But we are worried and we would like to find an explanation.
You say that when immobilization reduces mobility there are less binding opportunities. Please let me ask to you of you know any paper which discuss this reduction in binding opportunities caused by a decrease in ligand mobility.
Thank you very much in advance, your comments are very helpful
It is hard to find publication on this matter but the following four will give some background.
Edwards, P. R., Gill, A., Pollard, Knight DV, Hoare, M., Buckle, P. E., Lowe, P. A., and Leatherbarrow, R. J. Kinetics of protein-protein interactions at the surface of an optical biosensor Analytical Biochemistry 231: 210-217 (10-10-1995).
Edwards, P. R. and Leatherbarrow, R. J. Determination of association rate constants by an optical biosensor using initial rate analysis Analytical Biochemistry 246: 1-6 (1-3-1997).
Kortt, A. A. et al; Nonspecific amine immobilization of ligand can Be a potential source of error in BIAcore binding experiments and may reduce binding affinities; Anal.Biochem.; (253): 103-111; 1997
Johnsson, B., Lofas, S., and Lindquist, G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors Analytical Biochemistry 198: 268-277 (1-11-1991).