Choosing the Best Substrate for your ELISA Assay

Compare ImmunoChemistry Technologies' range of ELISA substrates and stop solutions to identify the best match for your enzyme system, assay format, and sensitivity requirements, all designed to deliver consistent performance and meet your specific assay needs.

Which substrate should I use?

There are a wide range of commercially available ELISA substrates, and selecting the best one for your specific ELISA is not always easy.

The starting point is to decide which signal-generating enzyme you wish to use - typically horseradish peroxidase (HRP) or alkaline phosphatase (AP) (read about our Conjugates and Stabilizers).

The signal generating enzyme is conjugated to a detection antibody in both antigen-down and antibody sandwich ELISA formats (view our Guide to Developing Optimized ELISAs and Conjugates and Stabilizers).

We discuss the merits of HRP and AP as signal-generating enzymes in our blog post Choosing the Right Conjugates and Stabilizers.

Signal-generating enzyme

The two most commonly used signal-generating enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP).

Colorimetric, fluorescent and chemiluminescent readouts are possible with HRP and AP. However, we will focus on colorimetric readouts as they allow easy visual monitoring of the reaction and can be quantitated using absorbance plate readers that are standard equipment in most laboratories.

Colorimetric substrates work by converting a colorless substrate to a colored product.

HRP does this in a redox reaction involving H₂O₂ as a co-substrate (for a detailed explanation of HRP chemistry, please take a look at HRP Redox Reaction Driven TMB Color Development).

AP works by hydrolyzing the phosphate group from a colorless substrate to generate a colored product.

Once you have decided which signal-generating enzyme you are going to use, the next step is to select your substrate.

Horseradish Peroxidase Substrates

ICT offers two different HRP substrates - TMB and ABTS.

TMB as a substrate

ICT offers TMB (3,3',5,5'-tetramethylbenzedine) as a substrate in 3 different formulations, each containing TMB and H₂O₂ in a ready to use solution. TMB is oxidized by HRP, using H₂O₂ as a co-substrate, to produce a soluble blue-green reaction product which can be read at either 370 nm or at 620-650 nm. Addition of a stop solution (see below) causes the chromogen to turn yellow, which can be read at 450 nm. The addition of stop solution also results in up to a 3-fold increase in absorbance value. The yellow color of the stopped reaction is stable for 1 hour.

Which TMB formulation is best for my ELISA?

• Looking for a general purpose TMB-based HRP substrate?
  TMB 1-Component HRP Microwell Substrate (SUBT) is our most popular substrate. This reagent is ideal when your target analyte is present in samples at concentrations in the ng-pg/mL range. It is a great reagent to use during ELISA assay development when the required assay sensitivity is still being evaluated.
  
  
• Need a very sensitive TMB substrate?
  TMB Super Sensitive 1-Component HRP Microwell Substrate (SUBS) is 40 times more sensitive than our SUBT. It is particularly useful when the level of target analyte is very low or when samples must be highly diluted (e.g. 1:10,000) to avoid sample matrix interference. It is also the substrate of choice with low affinity antibodies. Its superior sensitivity can also be utilized to reduce the assay incubation time.
  
  
• Samples contain high levels of the target analyte?
  TMB Slow Kinetic 1-Component HRP Microwell Substrate (SUBK) is approximately 25% less sensitive than our SUBT. It is ideal when your samples contain high levels of the target analyte - typically in the ug-ng/mL concentration range. It is also good for ELISA assays with long (e.g. overnight) incubation periods.
  
  Also consider ABTS 1-Component HRP Microwell Substrate (SUBA) - see ABTS substrates below for more details.

Stop Solution for TMB Substrates

All of our TMB substrates can be stopped using our Stop Solution for TMB Microwell Substrates (STOPT) when running ELISAs in end-point mode. The acidic stop solution causes the HRP-oxidized TMB substrate to turn from blue-green to yellow and the absorbance to increase as much as 3-fold. The yellow color can be quantitated at 450 nm in absorbance mode. The stop solution stabilizes the yellow color for 1 hour.

ABTS as a substrate

ICT also offers the alternative HRP substrate ABTS (2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonic acid) in a ready to use solution containing H₂O₂. ABTS is oxidized more slowly than TMB and so results in a less sensitive assay - which can be advantageous in some situation (see below). ABTS is oxidized to produce a blue-green product that can be read at 405-410 nm. If you wish to run your ELISA in end-point mode, simply stop the reaction by adding an equal volume of 1% sodium dodecyl sulfate (SDS). Unlike TMB, termination of the reaction with 1% SDS does not result in a color change or an increase in absorbance.

ABTS 1-Component HRP Microwell Substrate (SUBA) is ideal for samples containing high levels of the target analyte (ug-ng/mL). It is also a good substrate if you want to use lower sample dilutions (1:50-1:500). The lower sensitivity and slower oxidation of ABTS compared to TMB can be advantageous if you are experiencing high background with more sensitive substrates.

Alkaline phosphatase substrates

pNPP 1-Component AP Microwell Substrate with Stabilizing Pellets (SUBP) is a ready to use reagent formulated with stabilizing pellets for long term stability. This reagent contains the chromogenic AP substrate p-nitrophenyl phosphate which is hydrolyzed to generate the yellow-colored p-nitrophenol product which can be quantitated by measuring its absorbance at 405 nm.

The reaction can be terminated using our Stop Solution for AP Substrates (STOPP) which stops the AP reaction without causing any color or absorbance change. 

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