SureBlue TMB 1-Component Microwell Peroxidase Substrate for ELISAs From KPL

TMB (3,5,3’5’-tetramethylbenzidine) is a popular chromogenic substrate for the detection of the enzyme HRP (horseradish peroxidase), a commonly used antibody label for ELISAs (enzyme linked immunoadsorbent assay). An ELISA is a very specific analytic technique that uses an enzymatic system to amplify sensitivity. However, the amplification and development time vary with the enzyme and substrate used. TMB is one of the best and fastest amplifiers of the number of different substrates that can be used. The SureBlue TMB 1-Component Microwell Peroxidase Substrate from KPL provides a rapid detection method for HRP enzymes: 5-10 minutes to develop. Furthermore, if sensitivity is an issue, the signal is amplified by 2-3 fold once KPL’s TMB stop solution is added (see KPL’s Technical Guide for ELISA) Thus, chromogenic detection of HRP is much more rapid and sensitive than other substrates, such as ABTS and pNPP. My experience is with using the SureBlue TMB substrate in direct and indirect ELISAs in which the secondary antibody is conjugated with HRP.

In an ELISA, various substrates can be used to quantify the amount of protein, based on the amount of color generated, a factor that is directly proportional to the amount of the enzyme present. SureBlue TMB substrate is used with the enzyme HRP, typically conjugated to a secondary antibody. Once the HRP-conjugated secondary antibody is incubated with the primary antibody and its excess washed off, 100 ul of SureBlue TMB substrate is added to each well; color development occurs within minutes. The well will turn a blue color, sometimes instantly, upon the addition of the TMB substrate, or more commonly within 5-15 minutes. Then 50 ul of TMB stop solution is added to stop the color reaction from progressing; this also amplifies the signal by about 2-3 fold and turns the color to yellow. If the color change is dramatic, and for qualitative results, one may simply observe the color difference by eye to determine either a positive (bright yellow) or negative (colorless) reaction. For quantification, a plate reader is needed to measure the absorbance of each well at 450 nm. Each well has an absorbance that is compared to a standard curve on the same plate being read.

My experience with the SureBlue TMB substrate has been positive. The substrate has a rapid color development, sometimes immediately. Furthermore, when using other substrates such as the AP substrate, the result is much less amplified and takes much longer to develop. Even when an ELISA using an AP substrate was left overnight (24 hours) to incubate, the values were still lower than those obtained with the TMB substrate from KPL (see graph 1). Overall, this method is much less time consuming than AP and provides an amplified signal for samples that have a low concentration.

Graph 1. Comparing the enzymatic detection of pNPP (AP) and KPL’s TMB substrate (HRP) using the same primary antibody. Measured with a plate reader after 10 minutes for HRP and either 4 hours or 24 hours for AP.