Maximize immobilization level to minimize non-specific binding?

Hi all,

I'm trying to set up a capture assay, where I immobilize anti-his on a biacore CM5 chip. I then capture a his-tagged ligand, and flow an antibody as analyte. I'm having issues with non-specific binding (NSB), shown as binding to my reference flow cell, where no ligand is captured. 

One issue is that my interaction is low affinity and therefore I need to use very high analyte concentrations (up to 20 µM). When I reach such high concentrations, the NSB also becomes more of a problem. Does anyone know if such high antibody concentrations in general tends to result in stickiness/NSB to the CM5 chip?  

I have tried to minimize the NSB by adding NaCl to the running buffer. While this did lower the NSB, the affinity was also lowered to the level where I still couldn't get good data. Otherwise I'm using standard HBS-EP+ buffer as running buffer.

Is it possible to try to minimize the NSB by maximizing the immoblization level (to occupy more of the dextran matrix)? I have immobilized anti-his to approximately 7000 RU, and then captured the ligand to approximately 50 RU for kinetics analysis. But I could maintain the low capture level even with a higher immobilization level. Is there any chance of that working (assuming of course that the NSB is to the chip matrix)? And does anyone know what the maximum immobilization level on a CM5 chip is? Are there any drawbacks to high anti-his immobilization levels?

I will also try addition of BSA and carboxyl methyl dextran to my samples. And maybe try the CM4 chip instead. Has anyone managed to get rid of NSB by any or all of these suggestions? If so, what worked best?

Thank you!
/ A

Hi,
You have probably good reason to use an anti-his - his-ligand and antibody as analyte. Why not use the analyte antibody direct on the chip as a ligand?

That aside, go step by step. Is the nonspecific antibody binding high on a native surface? And is it lower on a deactivated surface? You can use something else to deactivate like ethylenediamine or PEG amine. Does it react with the capture antibody (check species cross reactivity)? If you suspect that the dextranmatrix is the reasen you can indeed try a CM4 or C1 or try the Sensor chip PEG as an alternative to dextran.

I think you can go to around 10000 RU on a CM5 chip but personally I don't think you can overcome NSB by adding more antibody to the dextran surface.

Regards
Arnoud