What is the ideal substrate for your ELISA analyses?

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…).