ELISA dilution effect - poor linearity (Jul/07/2010 )

General question:

In the past, I have observed poor dilutional linearity between sample dilutions. I am assuming there was something in the matrix that was interfering because as the sample was diluted out more, linearity improved. This would make sense except for the fact that spike recoveries were all good.

If there was something interfering in the matrix, wouldnt it affect the spike recovery?

Are there any other thoughts on poor dilutional linearity but good spike recovery?

Was your observation of the spike and recoveries made into the exact same samples that you serially diluted? Were your observations of the starting dilutions of the serial dilution made on the linear portion of the dose respose curve?

Yes, the sample was divided into two tubes, one spike and one unspike. both tubes were then serially diluted for a total of four dilutions. And yes, all values (spiked and unspiked) were above LLOQ and on linear portion of the curve.

Still confused

Thanks for the response!

If you do serial dilutions of the highest calibrator does it dilute in a linear fashion? I am guessing you are making your serial dilutions in your zero standard...

the poor dilution linearity...what is the deviation from predicted value?

sgt4boston on Jul 9 2010, 02:07 PM said:

The samples were diluted in assay diluent. There is no predicted value. I made up some data that might clarify my question. As you can see from the data (completely made up) below, sample linearity is reached at a 1:16 dilution. Adjusted results seem to stay constant. However, the spike recoveries are acceptable for all dilutions.

I have always been under the impression that sample dilutional linearity issues occur for primarily one of two reasons:
1.) Oversaturation of HCP i.e - not enough antibody to capture all of the HCP - Hook Effect
2.) Sample matrix interference

But if one of the reasons above is the case, why are the recoveries acceptable? If the problem was oversaturation of HCP or something in the matrix interfering, then my recovery should be very low.

Dilution Adjusted Result Spike recovery (%)
2 100 85
4 200 85
8 150 85
16 120 85
32 119 85
64 121 85



Again, thank you for your help.