ELISA Substrates: Overview & Applications

ELISA Substrates: Overview & Applications

ELISA Substrates: Overview

ELISA, an enzyme-linked immunosorbent assay, is an analytical plate-based biochemical immunoassay. It’s widely used in life sciences and biochemistry labs for the quantification of biomolecules, such as proteins, antibodies, peptides, glycoproteins, and hormones.  

The technique leverages the interaction between antigens and antibodies to produce measurable results. An antigen is a foreign protein that initiates an immune response, which can include a cascade of reactions after complexing with an antibody. An antibody is a protein produced by an organism’s immune system. The two types of antibodies involved in ELISA assay are primary antibodies and secondary antibodies.

In this technique, the antigens are immobilized on the surface of the ELISA plate followed by incubation with enzyme-conjugates antigen-specific antibodies. This leads to the formation of an antigen-antibody complex. The reaction product is then detected by incubating ELISA plates containing antigen-antibody complexes using a specific substrate and a suitable detection method.

The general ELISA protocol followed in labs includes the following steps:

  1. The antigens are immobilized on the surface of the ELISA plate.
  2. Using a blocking buffer the remaining parts of the plate are covered.
  3. ELISA plate with antigen is incubated antigen-specific antibodies at room temperature.
  4. Measure the reaction product or obtained signal using a plate reader.

The last step of the ELISA is the incubation of the ELISA plate, containing the reaction mixture, with a suitable substrate. The use of substrate is based on the detection method you use and enzyme-conjugated to antibodies, such as horseradish peroxidase (HRP), β-galactosidase, and alkaline phosphatase (AP). This helps in the signal amplification and visualization of the reaction occurring in the plate.

Some commonly used substrates used in ELISA applications are hydrogen peroxide, luminol, and P-Nitrophenyl-phosphate (pNPP substrate).  

In this article, we will review how these substrates work during ELISA assays, their functions, and their applications in different medical and biotech labs and industries.

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How Do ELISA Substrates Work?

ELISA substrates work by binding with the enzyme conjugated to detect antibodies used in the assay. The enzyme converts the substrate into a colored product or fluorescence or electrochemical signal, whose absorbance or reading is taken using a spectrophotometer,  spectrofluorometer, or electrochemical device for the quantification of the antigen in the given sample.

Different substrates yield different colors during their oxidizing reaction with the enzymes. For example, the horseradish peroxidase substrates produce the following results after reacting with the HRP enzyme:

  • ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt): Green
  • TMB (3,3′,5,5′-tetramethylbenzidine): Orange
  • OPD (o-phenylenediamine): Blue

The amount of color produced or the signal obtained is directly proportional to the amount of primary antibody that binds to the antigen immobilized on the surface of the 96-well plate.

Figure: An illustration of antigen-antibody complex formation and oxidation reaction between HRP enzyme and its substrate.

To obtain high-quality, consistent, and reliable protein measurement results. researchers today use ELISA kits. These assay kits are equipped with all the suitable reagents required to run a  specific ELISA assay. They are easy and quick to perform. Furthermore, they have the lowest inter- and intra-assay variability, high sensitivity, and highest possible lot-to-lot consistency.

There are also substrate kits available in the market that come with suitable enzyme-substrate reagent formulations for use at the detection step of ELISA. They make the process faster and easier.

Types of ELISA Substrates

Based on the detection methods they are utilized in, the ELISA substrates are classified into the following groups:

Chemiluminescent substrate

The light produced in reporter enzyme-substrate reactions is used as the signal for chemiluminescent detection systems. The chemiluminescent substrate solution used for the HRP enzyme includes luminol. The AP enzyme needs 1, 2-dioxetane-based reagents to produce a signal that is captured on X-ray film or charge-coupled device (CCD) cameras.

To increase the intensity, duration, and sensitivity of chemiluminescent signals, enhancer chemiluminescent substrates (ECL) are also used in the detection process. However, the dilutions of the substrate should be made to prevent false high-intensity signals.

Chromogenic substrate

These substrates react with reporter enzymes to produce colored insoluble reaction products that are detected using a spectrophotometer. The HRP substrates used in the method include 2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); 5-aminosalicylic acid (5-AS); 3,3’,5,5’-tetramethylbenzidine (TMB); and o-phenylenediamine (OPD).

The substrates for AP enzymes are 5-bromo-4-chloro-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT). The resulting colorimetric signal can be enhanced by using a stop solution to obtain a more accurate signal measurement.

Figure: An illustration of the mechanism of the chromogenic detection method.

Fluorogenic substrate

Fluorescent ELISA substrates require a fluorometer that can produce the correct excitation beam for fluorescent tags to emit signals. The fluorometers measure the signal through its excitation and emission filters.

The fluorogenic AP substrate used in the ELISA assay is 4-MUP (4-methylumbelliferyl phosphate), which produces a signal at an emission wavelength of 440 nm and an excitation wavelength of 360 nm. HPPA (3-p-hydroxyphenyl proprionic acid) and HPA (hydroxyphenylacetic acid) substrates are paired with the peroxidase enzyme.  

Figure: A representation of the fluorometric detection mechanism.

Choosing the appropriate substrate for your ELISA assay depends on the amount of sensitivity (lower or higher detection limit), the timing requirements, and the type of detection device to be required/used in the process.

What Are ELISA Substrates Used For?

ELISA substrates are crucial for the detection, amplification, and quantification of the signals obtained in the ELISA assay. They react with enzymes, producing colored products or signals that are measured using suitable devices.

ELISA Detection

ELISA is a biochemical assay used to identify and quantify biomolecules, such as proteins, peptides, glycoproteins, and hormones. The incubation of the ELISA plate, containing a reaction mixture of antigen-antibody (conjugated with a fluorophore or enzyme) complex, with a suitable substrate for reaction visualization is the last step of ELISA.

The substrates react with HRP or AP enzymes producing insoluble colored reaction products or signals, which are measured by the instruments, spectrophotometer, or spectrofluorometer.

What Industries Use ELISA Substrates?

HRP and AP are the two most commonly used enzymes used to conjugate antibodies in ELISA assay for the detection of antigens. It’s mainly because of their simplicity and high sensitivity.

In the assay, they react with suitable substrates, producing measurable signals for antigen analysis. Thus, ELISA substrates are one of the key components of ELISA protocols and are used in many different labs and industries.


Pharmaceutical industries use ELISA tests to detect a myriad of diseases, such as influenza, HIV, dengue fever, and Lyme diseases. Some ELISA kits are also available in the market to make the workflow faster and easier.

Further, ELISA assays are also crucial in vaccine development. They detect antibodies that are formed against deliberately injected antigens into the host.

Food Industry

In the food industry, ELISA assay is used to detect food allergens in the manufactured product, especially involving milk, almonds, peanuts, walnuts, and eggs. It prevents fraudulent substitutions in the food and proves their authenticity in the market.

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ELISA substrates are one of the crucial reagents used to carry out the ELISA workflow. They are of different types based on the enzyme used in the assay. For example, the type of substrate used against the HRP enzyme includes OPD, ABTS, and TMB substrate, whereas Alkaline phosphatase substrates include pNPP, BCIP, and NBT.

The substrates help in the visualization and measurement of the color or signal generated during the ELISA reaction.

It’s essential to use high-quality substrates during the reaction to reduce/eliminate background signals and obtain accurate and reliable results. And, this must be paired with high-throughput instruments to yield the right and consistent results.

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