ELISA Substrates

For your ELISA workflow needs, Sigma-Aldrich offers several options for substrates to detect horseradish peroxidase (HRP). These substrates differ in sensitivity, and you can select the appropriate substrate

TMB (3,3′,5,5′-tetramethylbenzidine) gives blue reaction products upon reaction with HRP. The products have major absorbance peaks at 370 nm and 652 nm. Addition of either sulfuric acid or phosphoric acid to those solutions changes the solution color to yellow, with an absorbance maximum at 450 nm. The advantage of TMB is that it is highly sensitive. However, it can produce significant background signal in the presence of excess protein or antibody. In addition, TMB is oxidized more quickly compared to other HRP substrates, which causes faster color development.

OPD (o-phenylenediamine) gives a yellow-orange, water-soluble reaction product with an absorbance maximum of 492 nm upon reaction with HRP. Sigma offers OPD in both tablet and powder forms. Our multiple OPD tablet offerings, with different amounts of substrate, allow you to tailor your protocol to the optimal scale for your needs. Solutions of OPS are readily prepared in buffered hydrogen peroxide solution.

ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]) gives a green, water-soluble end reaction product upon reaction with HRP. This end product gives two major absorbance peaks at 410 nm and 650 nm. Compared to OPD and TMB, ABTS is less sensitive for HRP detection. Color development is relatively slow, on the order of 20 minutes. This may provide advantages compared to OPD or TMB, if background is too high from using the more sensitive OPD or TMB substrates. Sigma offers ABTS available in different formulations: powder, tablet, or ready-to-use solution.

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A3219 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Liquid Substrate System
P6662 o-Phenylenediamine dihydrochloride tablet, 1 mg substrate per tablet
P6787 o-Phenylenediamine dihydrochloride tablet, 2 mg substrate per tablet
P8806 o-Phenylenediamine dihydrochloride tablet, 3 mg substrate per tablet
P8787 o-Phenylenediamine dihydrochloride tablet, 4 mg substrate per tablet
P3804 o-Phenylenediamine dihydrochloride tablet, 5 mg substrate per tablet
P6912 o-Phenylenediamine dihydrochloride tablet, 5 mg substrate per tablet
P8287 o-Phenylenediamine dihydrochloride tablet, 10 mg substrate per tablet
P4664 o-Phenylenediamine dihydrochloride tablet, 15 mg substrate per tablet
P7288 o-Phenylenediamine dihydrochloride tablet, 20 mg substrate per tablet
P8412 o-Phenylenediamine dihydrochloride tablet, 30 mg substrate per tablet
P1063 o-Phenylenediamine dihydrochloride tablet, 60 mg substrate per tablet
P9187 SIGMAFAST OPD tablet
S5689 Stop Reagent for TMB Substrate solid, ELISA, 650 nm
S5814 Stop Reagent for TMB Substrate powder, ELISA, 450 nm
T4319 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate, Slow Kinetic Form, for ELISA peroxidase substrate
T4444 3, 3′,5 ,5′-Tetramethylbenzidine Liquid Substrate, Supersensitive, for ELISA ready to use solution
T5569 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate, Super Slow, for ELISA peroxidase substrate
T8665 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System peroxidase substrate
T0440 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA peroxidase substrate