plasma interference - (Oct/19/2008 )

Dear Forumers,

I am having problem to detect IL-1ra in plasma.....
I suspect this is due to presence of some inhibotor in plasma.
Can someone help me with this?


Thanks
oche

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What exactly is it that makes you suspect inhibition? What kind of an assay are you using? Interleukins exist in very low concentrations in plasma and can be a bit tricky to detect. You might have to use some kind of signal amplification for this kind of detection.

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If your system is optimized and you do suspect an 'inhibitor' do Google search on heterophile antibodies.

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Hi SatanClaus,

I bought the kit from R&D and it come with a pair of Antibody and quantitative standard.

I suspect plasma inhibition becuase i spike my standard into plasma..... the OD decrease drastically.

any explaination for this?

Oche

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Possibilities include:
1. Matrix effect (serum/plasma v buffer)
2. Heterophile abs

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I have to agree. It sounds like a matrix effect or heterophile antibodies interfering in your assay.

If it is a matrix effect and you have plenty of the standard you used to spike your samples, you could try using the standard addition method. If you add known amounts of standard to your samples, and the effect is constant you can calculate your original concentration from your ELISA results. Wikipedia has a short entry for the technique, but if you can find the text book they cite (Harris's Quantitative Chemical Analysis) there is a fairly thorough section on how to do it and how to calculate the results (and the resulting accuracy, if my memory serves me correctly).

If the effect stems from heterophile antibodies you could either just compensate by adding more of the detection antibody, or you could try removing them by using whole sera. However, since heterophile antibodies compete badly it seems less likely that they could quench the signal in your assay by displacing the interleukins (they mostly give a false positive signal). Wouldn't rule this one out though. Those pesky little antibodies work in all kinds of weird ways.

This was what I could think of off the top of my head. Let us know if any of it helps.

Best of luck!

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Different approach:

Matrix effect...pre-dilute your samples 1:10 in your assay buffer this will help (providing your concentration is sufficient and you don't go below your assay sensitivity.

The assay should be optimized for the matrix you are testing. The manufacturer should have this info in their package insert.

Finally adding more detection ab or serum will not work for heterophile abs Best route is to pre-dilute the sample as above and also pre-incubate them with same species of abs used in the test system. The titer of heterophile ab can exceed 1:100,00+. After off line pre-incubation handle the sample as you normall would. Account for the dilution when you analyze your results.


I think you basically may have combination of both of these taking place.

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