There are a wide variety of ELISA substrates designed for different systems and detection methods. These ELISA substrates are designed to detect alkaline phosphatase (AP) and horseradish peroxidase (HRP). In addition to their sensitivity, other factors, such as cost and ease of use, may also factor in. Choosing the right ELISA substrates begins with determining the limits of detection required. From there, a general type of ELISA substrate can be selected and the specific substrate can be narrowed down. There are five major types of substrate available: PNPP, ABTS, OPD, TMB, and ONGP. Of these, TMB is one of the most versatile.
PNPP (p-Nitrophenyl Phosphate, Disodium Salt)
PNPP substrate is one of the most commonly and generally used substrate types. PNPP is used to identify alkaline phosphatase when assaying. This substrate is available as a ready-to-use product, powder, and in tablet form. All forms of PNPP will create a water-soluble reaction product, which will be colored yellow. PNPP substrate has a detection limit of 100ng/mL, primary antibody dilution of 1:500, and secondary antibody dilution of 1:5k to 1:20k.
ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt)
ABTS substrate provides a water-soluble reaction product that is colored green. It is commonly used to detect HRP and is available in either a ready-to-use solution or a tablet form. ABTS is not as sensitive as OPD or TMB and it may take some time to properly develop its color. Consequently, ABTS is not as often used as either OPD or TMB. ABTS substrate has a detection limit of 2.5ng/mL, a primary antibody dilution of 1:1K, and a secondary antibody dilution of 1:5K to 1:50K.
OPD (o-phenylenediamine dihydrochloride)
OPD substrate is available in either a powder or a tablet, though it is not available in ready-to-use formats such as ABTS and PNPP. OPD is used to detect HRP and produces a similar solution to PNPP, except in a yellow-orange color rather than a purely yellow one. OPD substrate has a minimum detection limit of 70pg/mL, a primary antibody dilution of 1:1K, and a secondary antibody dilution of 1:5K to 1:50K.
TMB (3,3',5,5'-tetramethylbenzidine)
TMB is one of the most versatile forms of substrate. It is often used for HRP detection because it develops faster than other substrates, though it is also far more sensitive. Care must be used when using TMB to ensure that the results are accurate. TMB comes in a variety of formats: slow TMB, Turbo TMB, Ultra TMB, and Novex TMB. The general TMB substrate is a 2-part kit that has a minimum detection limit of 60pg/mL. It produces a blue water-soluble reaction product which may then turn to yellow with the addition of either phosphoric or sulfuric acid. Ultra TMB has a detection limit of 20pg/mL, while slow TMB has a detection limit of 80pg/mL. With a TMB substrate, it's advisable to find the substrate that has only the detection limits strictly needed, as a "better" detection limit may also yield more sensitive results.
ONGP (ortho-Nitrophenyl-β-galactoside)
ONGP is not as commonly used as the other types of substrate. Available in a powder, ONGP substrate has a minimum detection of 100ng/mL, primary antibody dilution of 1:500, and a secondary dilution of 1:5K. ONGP is preferred for applications that require b-Gal as a reporter enzyme, and it will yield a water-soluble reaction product that is yellow in color.
Choosing the right substrate naturally begins by selecting the enzyme that is being tested for and the detection limits that are necessary for proper assay. In general, the substrate that is chosen should be the one that meets the minimum detection limits. Other factors, such as cost, may also factor in. In addition to choosing the type of substrate, it may often be necessary to choose how the substrate is presented: powdered, crystallized, ready-to-use, or in tablet form. This can make a difference regarding how easy it is to use on a practical level.
