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Elisa troubleshooting problem

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Old 09-28-2011, 12:32 PM
Pipette Filler
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Default Elisa troubleshooting problem



I am developing an inhibition/competition elisa,

Method:
Coat plate with antigen (diluted in coating) (plate A)
In different plate incubate monoclonal mouse ab with sample or standard (plate B)
Block (1%BSA,0.05%tween)coated plate (A)
Transfer contents of plate B to plate A, incubate
wash
Add hrp-anti mouse(diluted in block), incubate
wash
Add tmb


however my problem is that I think I have matrix effect, but when I try to dilute my sample I go beneath the sensitivity of the assay, any suggestions?

So far I've tried...
-decreasing mouse mono antibody - background* does not decrease much, sensitivity of the assay decreases.
-diluting sample in block buffer (1%bsa, 0.05%tween) - background decreases but the value is below linear part of standard curve (and as it is a competition elisa %b/b0 is ~100%)
-the competition is for antigen bound to the plate, I've tried reducing the concentration of this antigen, - not much effect seen on sensitivity, signal decreases.
-fiddled with lots of different incubation temps times - to no real effect

*by background I mean that when I omit the antigen coating but run the assay I get pretty much the same signal as when antigen coating is present. Running the assay with no sample present but still both antibodies included I get a very low signal, therefore its not lack of blocking.

Suggestions much appreciated!
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competition , elisa , matrix effect , problem , sensitivity , troubleshooting


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