When to stop ELISA enzyme reaction

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Jansen
Jansen's picture
When to stop ELISA enzyme reaction

Hi all,

I'm using TMB substrate for ELISA.

I found the following statement in The ELISA Guidebook but cant understand properly
"Stopping is usually done at a time when the relationship between the enzyme–substrate–product is in the
linear phase."

OD readings of different time points for 3 samples as attached.

Sami Tuomivaara
Sami Tuomivaara's picture
Jansen,

Jansen,

The only way to quantitatively compare enzyme activities (for example in different ELISA wells) is to assume that the absorbances in the samples have been increasing linearly with time to that point in time. This holds for kinetic enzyme analyses as well as ELISA. Generally the absorbance curves are linear until the substrate becomes limiting factor (unless there are other things happening like absorbance becomes so high that detector is saturated, or the enzyme starts losing activity, but these are not of concern here I think).

I think the plateau in your curves indicates that you are running out of substrate. You should decrease amount of immobilized sample, or dilute your antibodies more, and then measure the absorbance again. The bottom line is, whatever absorbance you use in quantification, make sure that the absorbance increase up to that point has been linear...

Cheers,

Jansen
Jansen's picture
 Hi Suola,

 Hi Suola,

Thanks for the explanation. May i know why BSA for blocking solution should be fatty acid free? I have tried to google around but can't find the answer.

Regards,
Jansen