Posted on Thu, Jul 04, 2019

Enzyme Immunoassay

An enzyme immunoassay is used to detect the presence of a target protein in a biological sample. A more common name for an enzyme immunoassay is the enzyme linked immunosorbent assay (ELISA). These assays are the cornerstone of most established clinical tests, and are typically the gold star diagnostic method. The ELISA is also used in many research environments because of its reliability and sensitivity. It is commonly able to detect protein down to concentrations as low as single picograms per milliliter. The sensitivity depends on the antibody-antigen pair and on the performance of the enzyme-substrate pair. Enzyme immunoassays are labeled assays because they don’t detect a protein binding event (between antibody and antigen) directly. Instead, these assays rely on an enzymatic reaction between an enzyme-conjugated label and a substrate to produce a colorimetric, chemiluminiscent, or fluorescent product. These products are then detected via a spectrophotometer or fluorometer and compared to a standard curve to produce quantitative concentration information about the target protein in the experimental sample.  

Free PDF guide:  "The Basic Guide for the use of Magnetic Bead   in ChemiLuminiscent ImmunoAssays (CLIA)" 

There are many techniques for performing an ELISA, but the main idea is that it is a buildup of antibody and antigen onto the bottom of each well of a 96-well plate. The configuration of this buildup is dependent on the goal of the assay, whether it is a direct or indirect assay, or whether the goal is to detect antibody or antigen. These details are addressed in a previous blog post (competitive ELISA from last month). Here we are focused on the interaction of the enzyme-substrate pairs. There are many different combinations used for these assays, and below is a list taken from a major supplier’s website. The overall theme is that there are two very common enzymes used in enzyme immunoassays: Horse Radish Peroxidase (HRP) and alkaline phosphatase (AP). The product produced is dependent on the substrate used and whether or not there are any signal enhancers added to increase signal intensity. Many enzyme-substrate pairs are marketed to provide enhanced detection in order to reliably detect protein concentration down to single pg/mL levels. The key is to produce an intense product that is easily detected over the background. 

Types of Enzyme-substrate pairs used for enzyme immunoassays


  • PNPP (p-nitrophenyl phosphate, disodium salt) substrate for AP that produces a yellow product which absorbs 405 nm light
  • ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt) substrate for HRP that produces a green product shich absorbs 410 nm and 650 nm light 
  • OPD (o-phenylenediamine dihydrochloride) substrate for HRP that produces a yellow-orange product which absorbs 492nm light. This substrate is enhanced by the presence of hydrogen peroxide 
  • TMB (3,3’5,5’-tetramethylbenzidine) substrate for HRP that produces a blue light that absorbs 370 nm and 652 nm light. 



  • CSPD and CDP-star substrate for AP 
  • Dynalight substrate with rapid glow enhancer substrate for AP. The enhancer is a polymer
  • Supersignal ELISA pico and femto chemiluminescent substrates for HRP are claimed to be extremely sensitive for low protein concentration detection 


Fluorescent or chemifluorescent: 

  • Quantablu fluorogenic substrate for HRP produces a fluorescent reaction product with absorption at 325nm and emission at 420nm. 
  • QuantaRed Enhanced Chemifluorescent substrate for HRP with A570nm/E585nm.  Good for avoiding autofluroescence from biological samples
  • Amplex Red reagent for peroxidase, which is a good general purpose substrate


There are plenty of other enzyme-substrate pairs available for enzyme immunoassays. Often, these will come in a kit with plates that are pre-bound with capture proteins. As always, the abundance of options is useful only if one spends time to completely understand the goal of the experiment and to choose appropriate assay conditions to produce meaningful and easily interpretable results.

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