Assay Substrates for ELISA Testing

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The substrate medium is an occasionally overlooked variable in an ELISA test kit. PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is the most commonly used but, depending on the application, it could prove advantageous to use a more specific substrate. Substrate mediums are used for different systems of testing, as well as various testing methods. Specifically, they are engineered to detect horseradish peroxidase (HRP) and alkaline phosphatase (AP). Things like substrate sensitivity and factors such as cost and ease of operation can influence how a substrate is chosen. 

Determining the limits of detection required is the first step to choosing a substrate for ELISA testing. Once a general substrate type is chosen, the process for determining the specific substrate can begin. There are five key substrates from which to choose: ABTS, OPD, PNPP, TMB, and ONGP. TMB is widely considered the most versatile for testing but comes with a caveat. Below is a list of these substrates and their most common use.

ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) 

The ABTS substrate is not considered to be as sensitive as OPD or TMB and therefore is not used as often. ABTS is available as a ready-for-use product or in tablet form. This substrate is used to detect HRP, but due to the reduced sensitivity, testing time takes longer to develop its signature green color.

OPD (o-phenylenediamine dihydrochloride) 

OPD is only available in powder or tablet form and is commonly used to detect HRP. The water-soluble reaction will cause the mixture to turn yellow-orange in color. 

PNPP (p-Nitrophenyl Phosphate, Disodium Salt) 

PNPP is used to identify alkaline phosphatase and is among the most commonly used substrate types utilized in ELISA testing. PNPP is a ready-for-use product that is also available in powder or tablet form. Every form of the substrate will cause a water-soluble reaction, turning the mixture yellow.

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TMB (3,3′,5,5′-tetramethylbenzidine) 

The TMB substrate is considered to be one of the most adaptable forms of substrate, but caution is advised regarding accuracy when using it for testing, as it is far more sensitive than other substrates and develops faster. Unlike the other substrates, TMB has several formats, ranging down from Novex TMB to Ultra TMB, Turbo TMB, and finally Slow TMB. The general TMB substrate comes as a kit divided into two parts and causes a blue water-soluble reaction. If sulfuric or phosphoric acid is added, then the reaction turns yellow. It’s advised that when using TMB, to use only the substrate within the detection limits that are strictly necessary, as attempting to use a type that may be determined to yield a better reaction could also produce a more sensitive result, putting the accuracy of the test at risk. 

ONGP (ortho-Nitrophenyl-β-galactoside) 

The ONGP substrate is not as commonly used as the other listed types of substrates. ONGP is generally chosen for tests that require b-Gal as a reporter enzyme and comes only in powder form. ONGP yields a yellow water-soluble reaction.

Choosing the correct substrate starts with determining the detection limit necessary and identifying the enzyme for which to test. The general rule for determining detection limits is to select the minimum limit that meets requirements. The cost of the test may become a factor, as well as the presentation of the substrate upon completion of the test. On a practical level, it may be easier to present the end result as either crystallized, ready-for-use, tablet, or powdered form, depending on your needs.

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