Substrate
Definition
The compound converted by the reporter enzyme into a coloured product. For HRP, 3,3',5,5'-tetramethylbenzidine (TMB) is the standard substrate, producing a blue product that turns yellow after the stop reagent is added. The intensity of colour is proportional to enzyme activity, which is proportional to the amount of bound analyte.
- Role
- Compound converted by the reporter enzyme
- Assay type
- ELISA
- Standard example
- TMB for HRP
- Reaction signal
- Blue product, turns yellow after stop reagent
- Relationship
- Colour intensity proportional to bound analyte
Common questions
Why is the stop reagent step necessary if the colour is already proportional to analyte amount?+
The enzyme keeps converting substrate as long as the reaction runs, so without a defined stop point the colour would keep changing and readings taken at different times would not be comparable. The stop reagent halts the enzymatic reaction and shifts the product colour, fixing the signal at a consistent endpoint for accurate measurement.
What happens to ELISA results if the substrate is contaminated or degraded?+
A degraded or contaminated substrate can produce weak, inconsistent, or false colour development even when the target analyte is present at the expected level, leading to unreliable readings. Laboratories control substrate quality and run known positive and negative controls specifically to catch this kind of failure.
Related terms
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- Cross-Reactivity
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- Enzyme Conjugate
- An antibody (or antigen) chemically linked to a reporter enzyme, most commonly horseradish peroxidase (HRP) or alkaline phosphatase (AP). The enzyme catalyses...
- Sandwich ELISA
- An ELISA format in which a capture antibody immobilised on the plate binds one epitope of the target antigen, and a separate...
- Solid Phase
- The inert surface to which an antigen or antibody is adsorbed at the start of an ELISA. Usually the well walls of...