Trusted by 10,000+ Scientists Since 2002. View Our Google Review, Selected Publications and 3,000+ Cited Publications at Google Scholar.

ELISA Substrates: A Complete Selection Guide

Jul 21st 2021

ELISA Substrates: A Complete Selection Guide

Enzyme-linked immunosorbent assay is one of the plate-based assay methods designed for quantifying and detecting proteins, peptides, hormones, and antibodies. In ELISA, the antigen goes through the immobilization process through a solid surface. Post that, it gets complexed with antibodies linked to specific enzymes. Moreover, one can successfully detect such a process by accessing the conjugated enzyme activities through incubation to produce a measurable product. One of the essential elements of this detection process is an incredibly antibody-antigen communication.

ELISA is typically performed in 384-well or 96-well polystyrene plates, which passively bind a protein and custom antibody. It is this immobilization and binding of reagents that allows a researcher to design ELISAs. Under the general procedure concerning ELISA, unless you utilize a plated kit with antibodies pre-coated on it, the ELISA starts coating the first layer, consisting of target antibodies and antigens. Moving on into the process, this gets absorbed into the 96-well polystyrene plate. Once you achieve this step, it is followed by blocking steps where several unbound sites enter the coating method with blocking agents.


Through a series of washes, one can incubate the plate by utilizing an enzyme-conjugated custom antibody. Besides, there are more series of washes that fall under this specific technique. The next step is to add the substrate that can produce a colorimetric signal. Finally, ELISA kits read the plate. Since the assay utilizes surface binding for separation purposes, many other washes adhere to the same methods in every ELISA step to eliminate unbound stock and material. You must remove the excess liquid during such a method to prevent the solutions from diluting into the next assay step.

The Importance of Uniformity

To ensure a significant amount of uniformity, ELISA kit manufacturers and pharmaceutical companies use specialized plate washers. ELISA kits can be relatively complicated and comprise several intervening steps, primarily while measuring protein concentration in heterogeneous samples like blood. However, one of the most tricky and varying steps in this process is detection. This is where different antibody layers can enter the formulation step to amplify signals.

This application talks about ELISA kits and three different detection systems- fluorescent, colorimetric, and luminescent. Regardless of the employed detection system, all of these need the immobilization of antigens or antibodies to a specific surface. Furthermore, they also need an appropriate enzyme label with a matching substrate suitable for its entirety. An enzyme-substrate reaction comprises several requirements, like development and timing conditions, that need optimization to produce an accurate, precise, and reproducible assay.

Colorimetric Assays

Such assays lead to colored reaction products that tend to absorb light under a visible range. The reaction product’s optical density is relatively professional to the analyte’s amount being measured. While opting for a suitable enzyme label, make sure that you utilize the following labels:

  • Horseradish peroxidase
  • Calf intestine alkaline phosphatase
  • E. coli ß-D-galactosidase

The mentioned enzymes are primarily utilized since they meet most of the criteria necessary to generate an inexpensive, sensitive, and easily performed assay. Such measures can consist of:

  • Stability at standard assay temperatures: 37°C, 4°C, and 25°C
  • Commercially available
  • Capable of going through the conjugation process against an antibody or antigen
  • Should be inexpensive
  • Must measure easily
  • Should consist of a higher substrate turnover figure
  • Shouldn’t be unaffected by the assay’s biological components

Selecting a Suitable Substrate

For every enzyme-linked immunoassay, the last process is the utilization and addition of an enzyme substrate. Many researchers choose a substrate for its quantitative yield of a fluorescent, colored, or luminescent reaction product. Besides, for a colorimetric assay, the rate of color development should be proportional, over a specific range, to the figure of enzyme conjugate available.

One must select the suitable substrate to administer the performing assay requirements. Meanwhile, substrates may generate either soluble or insoluble colored reaction products, where insoluble reaction products are ideal for membrane-specific assays like dot blots. ELISA kit manufacturers produce these dot blots at the reaction’s site.

Even though it is a visual and permanent record, the colored product’s intensity can be determined utilizing densitometry. But, insoluble products used at reaction sites are not efficient enough for solution immunoassays. Researchers state that substrates that become soluble products are ideal for ELISA. Both alkaline and peroxidase phosphatase have substrates that can lead to soluble colored products.

How do you determine the quality of a substrate? It primarily depends on the timing requirements, the sensitivity desired, and the detection device you will utilize. Several desirable substrates generate intensely colored reaction products at fast reaction figures for relatively sensitive assays. However, experts state that the most desirable assays need to go through a considerable dynamic range and substrates that allow the reaction product over an extended period of fifteen to thirty minutes.

Fluorescent Assays

ELFIA or Fluorescent immunoassays are a colorimetric variation of ELISA in which a conversion process occurs between an enzyme and a substrate. The fluorescence units or emitted light photons that get detected are usually proportional to the analyte amount that researchers measure in their tests and experiments. This is similar to those who work with gene synthesis. In contrast with colorimetric ELISAs, ELFIA is relatively more sensitive. It broadens the assay’s range and enables high readings to accurately measure as shown by the 2.0 to 4.0 OD limit.

Selecting A Suitable Enzyme Label

Three of the primary enzymes described here for colorimetric assays are similar to those used for ELFIA. But, their popularity’s order is different and varies. Alkaline phosphatase is one of the most widely utilized enzymes under fluorescent applications. Moreover, ELISA kit manufacturers and pharmaceutical companies utilize ß-galactosidase more frequently because of its significant theoretical sensitivity when combined with fluorogenic substrates.

Selecting a Suitable Substrate

Researchers opt for a fluorogenic substrate for its quantitative emission of light. Besides, it is essential to know that the light emission rate should be equivalent to the enzyme conjugate. Yet another factor is the room temperature of a suitable substrate. Its room temperature should be stable and exposed to standard room lighting.

Here, there are three main enzymes where every one of them comprises major fluorogenic substrates. Not only do researchers pair alkaline phosphatase with 4-methylumbelliferyl phosphate, but they also convert it to 4-methylumbelliferone through an emission wavelength of 440 nm and excitation wavelength of 360 nm. Moving on, they dissolve this substrate at 0.1 to 0.2 mg/mL in 1 mM MgCl2, pH 9.6, 100 mM diethanolamine.

Luminescent Assays

Like fluorescent immunoassays, luminescent assays are specific variations of standard ELISAs. Here, an enzyme converts substrates to reaction products that can emit light photons instead of producing visible colors. This assay is a product of an emission light that may lead to an electronically excited state. The multiple forms and types of luminescence like chemiluminescence, bioluminescence, and photoluminescence differ in their excited states. For instance, photoluminescence is fluorescence. The excitation process gets initiated with an emission light at a specific wavelength.

Selecting a Suitable Substrate

There are multiple factors while choosing the suitable substrate for an assay. Some of the elements are mentioned in the rundown.

  • Low background luminescence amid the ground state
  • Ability to produce intense light when in an active position
  • Ability to generate stable light emission over an extended period
  • Ability to generate stable light emission over an extended period

The substrate should have a stable form when kept at room temperature. Moreover, one should know that three of the most-utilized enzymes have several appropriate substrates. Besides, peroxidase comprises the most absorbed list of specific substrates like:

  1. Luminol
  2. Acridine esters and polyphenols
  3. Luciferin. T

TMB Substrates & Various Reagents 

HRP and various enzyme substrates concerning immunoassays are readily available in a wide range of sensitivities for other detection methods. Furthermore, specific substrates will perform better than others if you optimize them within a given detection limit or system. For instance, a chemiluminescent ELISA substrate with high sensitivity will be tricky to optimize in assays where microgram or nanogram protein amounts are being assayed.

Meanwhile, entry-level substrates may not produce a specific level of sensitivity needed to detect femtogram protein quantities. Even though ELISA kit manufacturers may require optimization to achieve ideal results with such an assay system, one may explore and research appropriate substrates and choose initial and secondary antibody dilutions.

Requirements for Selecting ELISA Substrates

ELISA substrates vary in ease-of-use, price, sensitivity (i.e., the lower detection limit), and their compatibility with imaging equipment. When it comes to more sensitive substrates, they cost more. As a result, it might be relatively natural to think that one must utilize affordable, sensitive substrates. However, this may not be necessarily correct since ease of use is an equally important consideration.

ELISA substrates need various procedures to prepare a specific substrate working solution and detect the signal. While some of them are ready to use, others require several materials. In addition, every substrate comprises distinctive requirements for optimization methods. In general terms, it is more challenging to determine and optimize ELISA protocols with sensitive substrates since the tiniest flaws in non-specific binding and blocking are detectable. So, opt for an ELISA substrate that may work slightly below the specified limits of detection needed for assays.

Colorimetric ELISA substrates

Many pharmaceutical organizations offer chromogenic substrate for ELISA production and development with AP or alkaline phosphatase and three other substrates for the HRP (horseradish peroxidase enzyme):

  • ABTS is utilized in the detection process of HRP and, therefore, yields water-soluble green end products. This green product comprises a couple of primary absorbance peaks - 650 nm and 410 nm.
  • TMB soluble substrates can yield a color close to blue while detecting horseradish peroxidase enzymes. One of the significant absorbance maxima or reaction products is 652 nm and 370 nm. Here, the color may change to the one close to yellow with phosphoric or sulfuric acid.
  • OPD is brought to use by researchers to detect HRP and yield water-soluble yellow-orange products through specific reactions. While ABTS had an absorbance peak of 650 and 410 nm, the OPD has an absorbance maximum of close to 490 nm.
  • PNPP is yet another popular substrate utilized to detect alkaline phosphatase within ELISA applications. This substrate produces a yellow water-soluble product that can absorb light at 403 nm.

Chemiluminescent ELISA substrates

  • CDP-Star and CSPD substrates two chemiluminescent alkaline phosphatase substrates. Not only are they ready to use for nucleic acid or protein but also solution-based assays.
  • With SuperSignal Chemiluminescent Substrate, it tends to provide a considerable performance for an extensive target protein range. It can be quickly optimized to recognize greater sensitivity than any entry-level colorimetric substrate.
  • DynaLight Substrate and RapidGlow Enhancer are also ready-to-use chemiluminescent substrate formulations that have gone through optimization processes to acquire enhanced results within the solution-based assay.
  • Yet another substrate is the SuperSignal ELISA Sensitivity Substrate which is one sensitive substrate readily available for various ELISA applications.

Fluorescent ELISA Substrates

  • QuantaBlu Fluorogenic is a prevalent substrate with a more extensive linear detection range and low-end linearity when it concerns fluorescent ELISA substrates. This stable reaction product comprises an Amax/Emax of 325/420 nm, allowing non-stopped, stopped, and kinetic assays to perform further.
  • Amplex Red Reagent is a highly stable and sensitive probe for H2O2. Since H2O2 can be placed and produced in several enzymatic reactions, researchers and labs may detect many different enzymes’ activities.
  • QuantaRed Enhanced Chemifluorescent Substrate is one of the sensitive fluorescent ELISA substrates readily available to detect HRP. Such a reaction product can only be stable for four hours if it consists of an Amax/Emax of 570/585 nm.
  • The last substrate is an Amplex UltraRed Reagent that can improve with Amplex Red reagent’s performance. It offers enhanced sensitivity and brighter fluorescence on a per-mole foundation in peroxidase or peroxidase-coupled enzyme assays.

The Bottom Line

Selecting a suitable substrate is equivalent to opting for the correct enzyme to test to detect specific limits necessary for a successful assay. In simpler terms, the chosen substrate should meet the minimum detection curbs or limitations. Other factors like cost are also essential to consider. While selecting a suitable substrate, it is necessary to opt for the one you can present:

Top