ELISA Assay - Saturated standard curve

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Cale
Cale's picture
ELISA Assay - Saturated standard curve

Hey,

I've been working on a problem that arose with an established ELISA. The assay was working well, but we ran out of substrate and I think since we started using a new one the the standard curve has become saturated at approximately 0.005 mg/L protein conc.
I've tried a few things:-
diluted HRP-conjugated antibody further (no effect except lower signal - still saturated) (have tried 1:10,000 - established, 1:50,000, 1:100,000, 1:200,000)
dilute primary further to 1:20,000 (no effect except lower signal)
increased concentration of primary to 1:5,000 (not much of an effect)
Also tried diluting the substrate (I know this isn't recommended)
Maybe the substrate is slightly different in that the HRP cannot convert it to product quite as quickly as the previous substrate? (but they are both TMB from same manufacturer :S)

Is maybe the best course of action to just try and dilute the samples so that they all fall below 0.005 mg/L?
What else might be causing this saturation effect?

Thanks for any help!
my other ideas to try: increasing conc of coating antibody (maybe not enough sticking to plate?)

ise702
ise702's picture
what is your detection system

what is your detection system?

Cale wrote:

Hey,

I've been working on a problem that arose with an established ELISA. The assay was working well, but we ran out of substrate and I think since we started using a new one the the standard curve has become saturated at approximately 0.005 mg/L protein conc.
I've tried a few things:-
diluted HRP-conjugated antibody further (no effect except lower signal - still saturated) (have tried 1:10,000 - established, 1:50,000, 1:100,000, 1:200,000)
dilute primary further to 1:20,000 (no effect except lower signal)
increased concentration of primary to 1:5,000 (not much of an effect)
Also tried diluting the substrate (I know this isn't recommended)
Maybe the substrate is slightly different in that the HRP cannot convert it to product quite as quickly as the previous substrate? (but they are both TMB from same manufacturer :S)

Is maybe the best course of action to just try and dilute the samples so that they all fall below 0.005 mg/L?
What else might be causing this saturation effect?

Thanks for any help!
my other ideas to try: increasing conc of coating antibody (maybe not enough sticking to plate?)

Cale
Cale's picture
In what respect? Are you

In what respect? Are you talking the plate reader? I'm using a Bio Rad 680 microplate reader set at 450 nm wavelength for detection. The antibodies are

coating : Anti-CRP (polyclonal)
primary: Anti-2 CRP Monoclonal clone c2 (mouse)
secondary: Dog anti mouse polyclonal (HRP conjugated)

TMB substrate

swannnyy
swannnyy's picture
What kinds of absorbance are

What kinds of absorbance are you talking about?
I'm sorry if I sound a bit dim, but just what doyou mean by saying your assay is saturated at 0.005 mg/l (5 ug/l? 5 uM?) What happens with the other standards?