Forum :: How to get the upper asymtote/limit of detection

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These are the posts of the old forum. It was not possible to transfer the user data, so they are missing in most of the posts. For new questions, go to the general discussions.

TOPIC: How to get the upper asymtote/limit of detection

How to get the upper asymtote/limit of detection 01 Apr 2009 02:00 #1


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+


How to get the upper asymtote/limit of detection 01 Apr 2009 02:00 #2

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?