TMB (3,3′,5,5′-Tetramethylbenzidine) has become the most popular colorimetric substrate for peroxidase (HRP) in various immunodetection analysis techniques due to its non- carcinogenicity (compared to OPD and ABTS,…) and its performance (detection sensitivity). However, optimal results require an optimized formulation according to the immunodetection technique (ELISA, Blotting, IHC) and an adapted protocol (temperature and incubation time, with or without stop, direct/sandwich ELISA).

What is the ELISA?

Applications and benefits of the TMB substrate

To this point, Uptima, the dedicated brand of Advion Interchim Scientific, provides several TMB solutions, 4 non-precipitating formulations for the major analysis technique that is ELISA (on microplate), as well as a precipitating formulation for blotting or IHC. You can find these different products at the end of the article.

All these formulations provide:

  • Performance: high detection sensitivity
  • Reliability: highly qualified and reproducible batches
  • Practicality: ready to use – Mono-Solution
  • Stability: from 24 months to 48 months depending on the formulation

In ELISA, TMB produces a slightly stronger signal in the blue than OPD (itself stronger than ABTS) in continuous kinetic mode as well as in stopped endpoint (data not shown). The addition of an acid that stops the HRP/TMB reaction increases this advantage tenfold by increasing the optical densities by a factor of 3 to 4. TMB is thus the colorimetric substrate of choice for the most sensitive assays and is favored by scientists for both routine and diagnostic use.

Comparative test of Uptima TMBs and competing TMBs

We’ve compared 4 Uptima TMBs to 5 competitors, in a direct ELISA protocol.


Figure 1: Direct ELISA with HRP-labeled rabbit IgG antibody revealed by 9 TMB substrates for 30 min at 37°C with acid stop (H2SO4). Optical density (OD) of wells measured at 450 nm is reported for the 1/120,000 dilution of ac. Negative controls (without ac-HRP) are all <0.053.


Uptima Friendly TMB #664700 gives the strongest signal of any TMB tested, allowing the use of antibodies at very high dilutions (here HRP-labeled rabbit antibody diluted 1/120,000), and provides maximum detection sensitivity with an unmatched signal-to-noise ratio, even better than our original, but still excellent, Standard TMB #66478. Like our TMB Wise #66477, it is also compliant with Undeclared Carriage and REACH XVII. For a guaranteed solvent-free (‘100% aqueous’) solution, you may prefer our TMB Aqueous #S0818 which has the same or better than its tested direct competitors (M3 and L).

For more information

The choice of an optimal substrate relies not only on the sensitivity of the substrate, its low background or its price!

For example, the stability of substrate batches, the dynamic range and the reproducibility of signals from one batch to others are important parameters, whether this is for R&D use or diagnostic or quality control. But beyond the choice of the TMB substrate, some operating procedures of the ELISA protocol, as in HTS screening, can improve the accuracy and robustness of the performed tests.

Contact our product manager to find the ideal TMB substrate for your application, from R&D to high-throughput testing, from manufacturing control to diagnostics. Our application specialists are also at your disposal for any optimization of your immunoassays (background noise; signal amplification; choice of substrate for your plate reader…).


Tips & Tricks

  • Background noise problem?
  • Improving the reproducibility of the ELISA?
    • Our reagent #UPS29590 (Stop Solution for TMB in ELISA ) improves the stability of the colored product formed, read from 405 to 450 nm, and thus the reproducibility of the signal in each analysis. TMB-Plate-Blue-Yellow(Stop)-Pink(Check)_Advion_Interchim_Scientifc_Blog_07/22
  • Checking for poorly distributed wells on your plates?
    • Try our TMB CHECK + #S0817: it will visualize, or through your PLC, any dispensing error.

Liste des produits ELISA

  • TMB substrats
    • TMB Blotting Substrate 1 Component, #295881 TechSheet
    • TMB ELISA Substrate Standard solution, #UP664780 TechSheet
    • TMB ELISA Substrate, AQUEOUS Solution, #UPS08180 TechSheet
    • TMB ELISA Substrate, “CHECK” Solution, #UPS08170 TechSheet
    • TMB Friendly ELISA Peroxidase Substrate Solution, #UP664700 TechSheet
    • TMB Wise ELISA Peroxidase Substrate, #UP664770 TechSheet
    • TMB Stop Solution (450nm reading), #S29590
    • TMB Stop Solution, 650nm reading, Liquid, #S51682 Tech Sheet
    • TMB Stop Solution, 450nm reading, Dry Blend Powder, #S2959C Tech Sheet
  • Saturants
    • BSA Solution 30%, Research Standard grade, #UP900102 Tech Sheet
    • Seablock ImmunoAssay Blocking/Saturating Agent, #UP40301A Tech Sheet
    • Seablock serum-free Blocking/Saturating Agent, #UPAP1370A Tech Sheet

Know More :