FcRn binding with NSB issues

Hello,
I've been trying to get an FcRn binding assay to work on a Biacore3000. I have been using the CAP chip kit to capture biotinylated FcRn and flow over antibodies of interest starting at 2000nM, with a 2-fold dilution series. FcRn binding antibody Fc is a pH dependent interaction, occurring at pH 6.0+/-0.2, typical regeneration is PBS pH 7.2-7.4. I have a couple of comparator antibodies that are well behaved, and then I have 2 antibodies of interest that have NSB issues. These 2 have been characterized as being hydrophobic.
I cannot add BSA into this experiment system, as FcRn also binds serum albumin, I cannot increase the pH. I have tried adding NSB blocker (dextran), increasing the NaCl slightly, running buffer PBS + 0.01% P20 (pH's 5.8, 6.0, 6.2, 6.4); running buffer HBS-EP pH'd to 5.8.
My method is to regenerate the CAP chip and apply the FcRn as ligand, then to run 7 concentrations of mAb + 0, each with a PBS pH 7.4 regen, the go back and regernate the CAP chip and add new FcRn (its a fragile molecule).
Do you have any suggestions as to what else I can try to eliminate the NSB? Is there another, non-BSA, carrier protein that might work?
I have also tried reversing the binding interaction by capturing the mAb with anti-kappaLC, but I am limited in the amount of FcRn, the FcRn stock is in 50% glycerol and I had problems matching buffer (buffer exchange on so little protein didn't seem feasible).
Below is an example of what I am calling a well behaved mAb, plus one of my problematic mAb's.

Thanks,
Cristina

Hi Christina,

A Hydrophobic interaction is difficult to counteract in an SPR system. Additives like glycerol, DMSO and dimethyl formamide have a high refractive index distorting the SPR signal. But you can try low concentrations in the running buffer and analyte dilution.
Also found a publication in which the authors measure the hydrophobicity with Triton-X100 (3) This detergent could be an alternative to P20. In addition other detergent can be used.

1. Giannetti, A. M., Koch, B. D. and Browner, M. F.; Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors. Journal of Medicinal Chemistry (51) 3: 574-580; 2008.
2. Ryan, A. J., Gray, N. M., Lowe, P. N., et al.; Effect of detergent on "promiscuous" inhibitors. J.Med.Chem. (46) 16: 3448-3451; 2003.
3. Yamaguchi, S., Mannen, T. and Nagamune, T.; Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor. Biotechnol.Lett. (26) 13: 1081-1086; 2004.

Kind regards
Arnoud