5 Various Types of ELISA Enzyme Substrate 

Enzyme-linked immunosorbent assay, commonly known as ELISA, is a plate-based assay method developed primarily for detecting and quantifying antibodies, proteins, hormones, and peptides. During ELISA, the antigen undergoes the process of immobilization via a solid surface. After this, it is


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Also, you can detect this process through access to the conjugated enzyme activities achieved with incubation to create a quantifiable product.  

Given the various processes required to reach a credible and viable result, producing a colorimetric signal through the addition of a substrate is perhaps the key determinant since it enables ELISA kits to read the plate. As such, researchers and scientists using ELISA must match the correct enzyme label to the appropriate substrate to get an accurate and reproducible assay.  

This post will discuss the five different types of ELISA enzyme-substrate used to obtain reproducible assays. But first, a bit of explanation.   

What is an ELISA Enzyme Substrate? 

ELISA substrates are vital components that are indispensable in any successful ELISA assay. To get the best possible detection, the chosen substrate needs to be extremely sensitive. You can obtain Different ELISA substrates for assays by purchasing from several suppliers, but first, you need to know the various types, which are as follows: 


Chemiluminescent Substrates 


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CDP-Star and CSPD are chemiluminescent 1,2-dioxetane alkaline phosphatase substrates that give off light with the highest light intensity of 475 nm. In other words, these are “glow” substrates that offer a reliable high signal for the duration of 15 to 60 minutes according to the temperature. Otherwise, you can boost the sensitivity of the piece by adding Emerald-II luminescence enhancers.  


Key substrate enhancers include the following:  

  • DynaLight substrate with rapidGlow enhancer - This is a chemiluminescent formulation that comes as a ready-to-use substrate optimized to yield quicker results in solution-based assays. It is typically categorized as a flash and glow substrate that offers sustained and early maximum signal as soon as 2-10 minutes. 
  • SuperSignal ELISA Femto maximum sensitivity substrate - It is among the most sensitive substrates you can use for ELISA applications. If optimized appropriately, it has a lower detection limit of 1 to 10 orders of magnitude below the average colorimetric substrates.  
  • SuperSignal ELISA Pico chemiluminescent substrate - It performs excellently, specifically for a wide variety of target protein amounts, and can rapidly identify with higher sensitivity than the typical colorimetric substrates.  


Chemiluminescent Substrates 

In simple terms, colorimetric ELISA enzyme-substrate reactions produce soluble products having an absorbance or optical density that’s measurable by a spectrophotometer. It’s easy to detect analyte concentration using colorimetric substrates because substrate color development depends solely on the concentration of the bound analyte.  

Several pharmaceutical companies provide chromogenic substrate for ELISA development and production with AP and three different substrates for HRP, namely:  


  • TMB soluble substrates - These can generate a pigmentation similar to blue while spotting HRP enzymes.  
  • ABTS - Is used in the detection step of HRP. As such, it produces end products that are green and water-soluble.  
  • OPD - This is a researcher produced and meant for detecting HRP by generating water-soluble yellow-orange products.  


Fluorescent Substrates 



These are the substrates that fall below fluorescent ELISA substrates: 

  • QuantaREad enhanced chemifluorescent substrate - It is the most sensitive fluorescent ELISA substrate used to detect HRP. Its reaction generates resorufin having 4-hour stability after the reaction stops.  
  • Amplex red reagent - It is a stable and extremely sensitive probe for H2O2. 
  • Amplex ultraRed reagent - This substrate enhances Amplex red reagent’s performance providing a more vivid fluorescence with improved sensitivity.  
  • QuantaBlu fluorogenic substrate - Possesses a wide linear detection range and detection linearity that’s low-end for HRP. 


TMB ELISA Substrates 

Tetramethylbenzidine, shortened as TMB, exists as a chromogenic substrate popularly used in several staining procedures during the HRP detection step and has become a widespread application in most ELISA procedures. If obtained from the right source and used correctly, it can provide adequate sensitivity and stability, longer shelf life, and an above-average signal generation rate. They normally come in ready-to-use formats, making them easy to incorporate into the manufacturing process of existing essays.  


p-Nitrophenyl Phosphate (pNPP) Substrates 


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This is a typical substrate for Alkaline Phosphatase (AP). It is abbreviated as pNPP; this substrate produces a yellow solution that can absorb light at 405 nm. It exists in a ready-to-use state, is the most convenient, and has widespread use in many ELISA assays. It can be used along with stabilizer pellets to serve as the preferred alternative for AP-based applications.  


You can compare choosing the perfect substrate to selecting the suitable enzyme to test or detect definite limits required for a successful assay. Simply put, the substrate you choose has to meet the minimum detection limitations or curbs. However, you also need to consider other factors, the chief of which is the cost of the substrate.  



Ellyse Warne

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