Cannot Find the Kd of Antibody

Recently I try to measure kinetic for some antibodies by using CM5 chip, immobilized the protein(ligand) on the sensor chip, and flow the antibody(analyte) over that. the buffer that I used was HBS EP+(GE) and the concentration of analyte was 333.33nM to 0.533nM, flow rate 50ul/min, the association time was 60 sec, and dissociation time 2400 sec.
The problem is, with that long dissociation time I still cannot find the Kd of the analyte. I have to try to change the buffer from HBS EP+ to PBS-T (0.05%tween20) or HBS EP+ which adjust NaCl from 150nM to 250nM, but still can not fond the Kd.
Do you have any suggestions to fix this problem?
Thank you

Hi,
A slow dissociation needs a very stable instrument, with minimal drift. The dissociation in the sensorgram has more positive drift than dissociation. To overcome this you should use the double referencing. When this is not enough you can use other approaches (see below).
To speed-up experiments with long dissociation times you can use the short & long approach ( www.sprpages.nl/experiments-2/strategy ).

If you want to use SPR, the best method to is probably a solution binding/affinity experiment (1).

A simpler alternative is what we call a solid state affinity binding via ELISA. Simple coat your receptor on the ELISA plate and incubate with the antibodies overnight (use an eleven point dilution series + zero antibody) to get a true equilibrium. Then wash the plate and detect the bound antibody with a reporter. Plot the colour reading against the antibody concentration and fit (equilibrium fit). This will give the KD.

1. Day, E. S., Capili, A. D., Borysenko, C. W., et al.; Determining the affinity and stoichiometry of interactions between unmodified proteins in solution using Biacore. Analytical Biochemistry (440) 1: 96-107; 2013.

Kind regards
Arnoud