The enzyme-linked immunosorbent assay (ELISA) is valuable for assessing and quantifying particular proteins in a complex mixture. It allows immobilized protein sample examination in microplate wells through the use of specific antibodies. Perlmann and Engvall initially described this method of protein analysis in 1971.
Researchers first render the target macromolecule on a solid surface before tagging it with an antibody connected to a reporter enzyme. Detection is achieved through the measurement of the reporter enzyme activity. This is achieved by incubating with a suitable substrate that results in a measurable product.
An essential aspect of the detection method is an exceedingly specific antibody-antigen reaction. Therefore, it’s necessary to know the appropriate substrates for a given system and detection method.
The Role of ELISA Substrates
There are several types of ELISA substrate meant for different applications of ELISA. The primary function of these substrates is the detection of alkaline phosphatase (AP) and horseradish peroxide (HRP). However, substrates vary in their sensitivity. Apart from this, other details such as ease of use and cost may impact their selection for use in a given detection method.
Nevertheless, due to the role substrates play, it’s essential to choose the right ELISA substrate. The process for selecting suitable ELISA substrates starts with understanding the limitations of the required detection. Following this, you can choose a general type of ELISA substrate and begin narrowing it down to help pick a specific one.
In terms of substrates for ELISA, there exist five distinct types, namely:
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ABTS
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ONGP
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OPD
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PNPP
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TMB
You can find the details about the functions of each group of ELISA substrates in the next section.
ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt)
The substrate called ABTS makes available a water-soluble reaction product with a visible green coloration. Its typical use is for the detection of Horseradish peroxide, and it exists in two forms. The first form is a tablet, and the second is a ready-to-use solution. Compared to the other ELISA types like TMB and OPD, ABTS isn’t as sensitive.
Therefore, the green color usually takes some time to develop properly. As a result, ABTS is not as frequently used as TMB or OPD. The detection limit of ABTS substrate is 2.5ng/mL. It has a 1:1K primary antibody dilution and between 1:5K to 1:50K secondary antibody dilution.
ONGP (ortho-Nitrophenyl-β-galactoside)
ONGP is also another substrate that doesn’t see widespread use as the others. It is available as a powder and has a 100ng/mL minimum detection. The primary antibody dilution for the ONGP substrate is 1:500, while its secondary dilution is 1:5K. Although it’s not commonly used in ELISA, it is thought preferable in applications in which b-Gal serves as a reporter enzyme. The water-soluble reaction product that this substrate produces has a yellow color.
PNPP (p-Nitrophenyl Phosphate, Disodium Salt)
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Among all the substrates used for ELISA, the p-Nitrophenyl Phosphate, Disodium Salt, is the most common since it serves more general purposes. It is primarily used to detect alkaline phosphatase during assaying. It is also one of the substrates that exist in a ready-to-use form and, in addition, tablet and powder forms.
Each of these forms of PNPP, when used, will produce a water-soluble reaction product with a yellow color. It comes with a detection limit of 100ng/mL. On the other hand, its primary antibody dilution sits at 1:500K, and secondary antibody dilution is between 1:5K to 1:20K.
OPD (o-phenylenediamine dihydrochloride)
The ELISA substrate called o-phenylenediamine dihydrochloride (OPD) also exists in two forms, namely tablet and powder. Even though it is not found in ready-to-use states like the ABTS and PNPP substrates, OPD is also valuable for identifying Horseradish peroxide.
Apart from this, the solution produced with this substrate is similar to that of PNPP. The only difference is that the resulting solution using OPD is yellow-orange instead of purely yellow. The minimum detection limit of OPD is 70pg/mL. Also, its primary dilution is 1:1K, and its secondary antibody dilution range is 1:5K to 1:50K.
TMB (3,3′,5,5′-tetramethylbenzidine)
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Of all the substrates, TMB is the most versatile one. It is mainly used in the detection of HRP due to its relatively faster reaction. Hence, TMB is much more sensitive than the other substrates. As such, its use requires extra care to ensure accuracy in the results. It’s available in diverse formats like Novex TMB, Ultra TMB, slow TMB, and Turbo TMB.
The typical TMB substrate comes in a 2-part kit having a 60pg/mL minimum detection limit. It yields a blue redaction product that is also water-soluble like the other substrates. This product may later change color to yellow when sulfuric or phosphoric acid is added to it. The detection limit of slow TMB is 80pg/mL and that of Ultra TMB is 20pg/mL.
Conclusion
Determining the appropriate substrate usually starts by choosing the enzyme under consideration and the detection limits required by the basic principles and types of ELISA. Generally speaking, the chosen substrate needs to fall within the minimum detection limits. Furthermore, it may be necessary to select the presentation of the substrate, whether it be ready-to-use, crystalized, tablet, or powdered form. All of these factors can influence accuracy.
