How to get the upper asymtote/limit of detection

Hi,

Here is my elaborate Biacore problem:

I'm doing a concentration analysis in which I run a standard and plot its curve using a 4 parameter fit. So far, everything has been going good but in order to validate this assay, it would be a lot easier to have a true sigmoidal standard curve in which I can assign the LLOD & the ULOD. Right now, I only have the lower limit of detection but I can't seem to reach my upper limit of detection no matter what I try. I've tried lowering dramatically my immobilization level (from ~9 000 RU to ~200 RU) and increasing my contact time (from 60 sec to 300 sec) but I still don't get the plateau at the top end of the curve like I would want. I am limited in the starting concentration of my standard because I am already starting close to a neat concentration so I cannot change that. I was wondering if anyone has any other ideas on how to manipulate the Biacore method so that I could reach my saturation level/ULOD?

P.S. I am currently testing different flow rates right now.

Here is some general info:

Biacore: T100
Chip: CM5
Buffer: HBS-EP+

Thanks!

For saturation of the ligand surface you need in general 50 - 100 x KD concentration and a long enough injection time to reach equilibrium. This is thus depending on the kinetics between the ligand and analyte.

Theoretical Req: Req = (ka.C / ka.C + kd) x Rmax (RU)
Time to equilibrium (95%): t95% = -ln(1-0.95) / ka.C + kd (s)

Does your analyte injection reach equilibrium during injection?
How is the actual Rmax or Req compared to the theorectical?
Does each higher analyte concentration still give a higher Req?

Arnoud