I am working with ELISA and use HRP conjugate as detection antibody and TMB substrate. All of the protocols I have used so far the TMB substrate reaction has been stopped by adding an acidic solution followed by reading the absorbance at 450 nm. No I have heard someone prefer stopping the reaction with some other stopping reagent (I don't know what that reagent contains) and read the absorbance at 650 nm. Does anyone here know why one would like to stop the reaction and read the absorbance without the colorshift from 650 nm to 450 nm?
It is not essential to stop the reaction (endpoint method), but it allows you to make a single measurement of each well, so it is simple. The alternative is the measure the rate of the reaction while it is happening. This requires reading the absorbance at several times and calculating the slope of the absorbance increase versus time. Many plate readers have this kinetic capability. I have found this to be a more sensitive method than the endpoint method.
Dear if you fail to stop the reaction at that stage the reaction will continue which you will notice with the intensity of the colour change and will definitely affect the result you will obtain.
The specific colors obtained with particular enzyme-chromophore systems at specific wavelengths in spectrophotometers reading.
Stopping Reagents are added to prevent further enzymatic reaction in ELISA. This is performed at a time as determined for the specific assay. Stopping is usually done at a time when the relationship between the enzyme–substrate–product is in the linear phase. Molar concentrations of strong acids or strong bases stop enzymic activity by quickly denaturing enzymes. Other stopping reagents are enzyme specific. Sodium azide is a potent inhibitor of HRP, whereas EDTA inhibits AP by the chelation of metal ion cofactors. Since the addition of stopping agents may alter the absorption spectrum of the product, the absorption peak must be known. For example, OPD/ELISAs stopped by sulfuric acid are read at 492 nm (450 nm before stopping). TMB /ELISA stopped by acid is read at 450 nm (stopped) and 650 nm (not stopped).
The addition of stopping agents can also increase the sensitivity of an ELISA. In the addition of stopping reagent, the volumes must be kept accurate, since photometric readings are affected if the total volume of reactants varies.
I understand how the enzyme works and how the stopping reagent works. What I wonder is why one would like to use a stopping reagent that does not cause a colorshift instead of stopping the reaction with an acid which changes the absorbance from 650 to 450? Does anyone know?
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