ELISA Enzyme Ready-To Use Substrates2018-01-29T14:30:11+00:00

Colorimetric Alkaline Phosphatase and Peroxidase Substrate ELISA Detection Systems


Enzymatic detection method

There are several different types of detection technologies that can be used: radiolabeling, fluorescent labeling or enzymatic labeling. The most commonly used enzymes are, as the detection system in ELISAs,  Horseradish peroxidase (HRP) and Alkaline phosphatase (AP). In order to be used in the assay, HRP and AP molecules must first be conjugated to the detection Ab or Ag. Consequently, the choice of several reagents depends on the enzyme.

AP- and HRP-conjugates can be detected and quantitated using chromogenic substrates that result in soluble, colored products when development takes place. The levels of the enzymatic reaction, and consequently the analyte’s concentration, are determined by monitoring the signal development at specific wavelengths. The most commonly used chromogenic ELISA substrates are 3,3′,5,5′-Tetramethylbenzidine (TMB) for HRP and p-NitroPhenyl Phosphate (pNPP) for AP.


Substrate type Specificity Reaction color Reaction read at Stopped reaction Stopped reaction read at
pNPP AP Yellow 405 nm Yellow 405 nm
TMB HRP Blue 620-655 nm Yellow 450 nm


Colorimetric HRP systems – Ready-To-Use TMB Substrates

Generally,- HRP systems are advantageous compared to other detection systems as both the enzyme conjugate and colorimetric substrates for the detection are economical attractive. The most commonly used colorimetric substrate for ELISA colorimetric HRP systems is Tetramethylbenzidine (TMB). Kem-En-Tec Diagnostics’ TMB solutions combine TMB, buffer and H2O2 forming a ready-to-use, chromogenic system that may be stopped with a weak concentrated sulphuric acid (down to 0,2M) resulting in a a 2-3 fold increase in sensitivity compared to the non-stopped absorbance reading. TMB Colorimetric Substrates from Kem-En-Tec Diagnostics provides the following characteristics and formulations:

  • Free of harmful organic solvents
  • Aqueous based
  • Ready-to-use
  • Negligible batch-to-batch variations
  • Recommended stopsolutions do not have hazardous acid concentrations

Prestained versions

All Kem-En-Tec Diagnostics’ TMB substrates are also available as unique color-coded products for traceable pipetting. The red indicator makes manual pipetting traceable, while in automatic analyzers, it offers a convenient tracking system for reagent supply. The color disappears after stopping and does not interfere with the assay.

TMB ONE an extremely stable and robust TMB substrate

TMB PLUS2 a cost effective improved version of Kem-En-Tec Diagnostics TMB PLUS

TMB X-tra 60% higher OD values compared to the sensitivity of TMB ONE

TMB SENS for high superior sensitivity and fastest assay development


NEW Products – more details below

TMB EASE Directly comparable to 2 mg OPD tablets

TMB X-tnd No Precipitation in high OD-Readings from Highly Positive Samples, sensitivity equal to TMB X-tra


Switch easily from OPD Tablets to Ready-to-Use TMB

The NEW TMB EASE was developed and fine-tuned to match exactly 2 mg OPD tablets. Alternatively, switching to our conventional TMB products, you have the opportunity to

  • Reduce incubation time
  • Dilute your conjugate further and hereby reduce assay cost




Compared sensitivity and kinetic curves of Kem-En-Tec Diagnostics’ TMB EASE. TMB EASE is compared directly to 2 mg OPD tablets as well 10 mg OPD tablets (same assay concentration used).


No TMB Precipitation of colored end product with High Positive Samples

The NEW TMB X-tnd is developed to support assays which require low detection limits while supporting OD readings of highly positive samples – and with NO precipitation of the chromogenic TMB substrate.

This Ready-To-Use TMB substrate will, like our other TMB substrates, be efficiently stopped with a low acid concentration, i. e. requiring NO hazardous labelling.



+5.5 OD-readings after 60 min show consistent OD values using the TMB X-tnd compared to main competitors.



Colorimetric AP systems – Ready-To-Use pNPP Substrates

In some cases HRP colorimetric detection systems face decreased sensitivity when compared to AP colorimetric detection systems. The most commonly used colorimetric substrate for ELISA colorimetric AP systems is p-NitroPhenyl Phosphate (pNPP)

Kem-En-Tec Diagnostics’s pNPP substrates are liquid buffer solutions containing stabilized pNPP. In the presence of AP, pNPP is hydrolyzed rapidly to p-nitrophenol and inorganic phosphate. The reaction can be stopped by adding an equal amount of 0,1 M sodium hydroxide (NaOH) solution and the intensity of the color is directly proportional to the concentration of the reactant.

Our ready-to-use mono-component substrates are stable up to 36 months from manufacture date when stored at 2-8°C, without loss of activity (for details see below).


Product characteristics:

  • Free of harmful organic solvents
  • Aqueous based
  • Ready-to-use liquid
  • Highly active and stable formulations
  • Negligible batch-to-batch variations



pNPP Kem-En-Tec Diagnostics most sensitive pNPP substrate

pNPP ONE Kem-En-Tec Diagnostics most robust pNPP substrate,- recommended in hot climate countries




In order to assist and inspire R&D employees, Kem-En-Tec Diagnostics has developed a comprehensive guide for ELISA Assay Development. The Guide takes the reader through the individual steps of an ELISA and provides Hint and Tips to each step.

The introduction presents the principle of the ELISA Assay, different basic types of ELISA Assays, modifications and combinations of these as well as describes the pro and cons of the different ELISA Assay protocols.

The last part includes an overview of suggestions to reagents with a view to optimize the ELISA Assay i.e. maximize ELISA Assay sensitivity, minimize ELISA Assay background and maximize ELISA Assay stability.

Please click to download our ELISA Guide

Environmental considerations during assay development

IVD manufacturers are working towards more environmentally conscious and sustainable manufacturing. Much of these efforts have been focusing on replacement of toxic chemicals for the protection of health of the employees. Kem-En-Tec Diagnostics supports these requirements, recommending ECO-friendly colorimetric substrates and reagents with no toxic preservative formulations.

Now also of highest priorities are reducing usage of facility water and energy use, reducing facility waste, increasing facility recycling, and sourcing recycled/recyclable product packaging. Kem-En-Tec Diagnostics´ Eco-sensible options are available for the development and production of diagnostics assays without compromising assay quality and stability. Further, those options can reduce production cost.



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