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Posted on Thu, May 25, 2023

Enzyme Immunoassay

Immunoassay tests are biochemical/bioanalytical methods that detect an “analyte” and quantify its concentration in a complex mixture of chemicals or biological fluids (e.g. serum or urine). Analytes can be a micro- or macromolecule (e.g. protein, nucleic acid, polysaccharide or lipid) or chemical substances (e.g. hormones, drugs).

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

Generally, immunoassay techniques utilize the specificity and affinity of an antigen/antibodies toward their complementary ligand (i.e. “analyte” can be either an antigen binding an antibody or vice versa), which generates an immunocomplex reaction detectable by using a reporter molecule called label. Based on the latter, there are "label-equipped immunoassays" and "label-free immunoassays".

"Labels" - the third component of immunoassay - are components chemically linked or conjugated to the antigen/antibody of interest and are detectable through various means by measuring their produced signal in response to the binding (e.g. emitting radiation, producing a color change in a solution, fluoresce under light, or light-induced emission). The majority of Immunoassays are label-equipped Immunoassays.

Label-free immunoassays, on the other hand, do not function with labels, but instead employ detection methods that do not require the modification or labeling of the components of the assay. Surface plasmon resonance is an example of technique that can detect binding between an unlabeled antibody and antigens.

Some immunoassay techniques often use a “calibrator” which is a solution containing the analyte with its known concentration. Comparison of an assay's response to a real sample against the assay's response produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.

Types of Immunoassays

On the basis of the nature of labeling compound, there are different types of Immunoassays:

  • Radioimmunoassay;
  • fluoroimmunoassay;
  • spin immunoassay;
  • luminescence-based assay;
  • enzyme immunoassay.


There are also other labels in use such as iron oxide, latex, red cells, nanosilica, nanomagnetic and metallic ions.

Enzyme Immunoassay

Enzyme Immunoassays are the most common labels, among all, to detect the analyte in question. Based on the assay, enzyme is coupled either to an antibody or antigen. The enzymes initially were employed to replace radioactive markers used in radioimmunoassays.There are different types of Enzyme Immunoassays:


Enzymes used in ELISAs include horseradish peroxidase (HRP), alkaline phosphatase (AP) or glucose oxidase. These enzymes allow for detection often because they produce an observable color change in the presence of certain reagents. In some cases these enzymes are exposed to reagents which cause them to produce light or chemiluminescence.

CLIA, among all, offer a faster analyte detection by employing the principles of “luminescence” - when a material transitions from an excited to a ground state, it emits light. The enzymes used in the chemiluminescent magnetic bead immunoassay convert a substrate to a reaction product, which generates a photon of light instead of producing a distinct color called luminescence.

Types of Enzyme Immunoassay

Based on separation criteria of immunocomplex reaction there are two types of Enzyme Immunoassays: homogenous and heterogenous.

Homogenous Immunoassay: in these assays, the enzyme coupled to an antigen/antibody retains its activity partially after the reaction. Hence, separation of the immunocomplex from the reaction mixture is not required for detection. The change in enzyme activity relates to the concentration of the analyte. Such assays are used mainly in the pharmaceutical industry and also are known as enzyme multiplied immunoassay technique. The homogenous method commonly is used for the measurement of small analytes like drugs. The absence of a separation step makes it an easier and faster method.

Heterogenous Immunoassay: separation of immunocomplex from the reactants is a prerequisite for analyte estimation. In such assays, also known as ELISAs, an antibody or antigen is bound either noncovalently or covalently to a solid matrix. The unreacted antigen or antibody is removed, and the bound count is taken. The solid matrix can be a microtiter plate, nitrocellulose membrane, polystyrene tubes or beads, nylon beads or tubes, or magnetic beads. The latter technology, among all, offers a simple, fast and efficient way of collecting the analyte-bound magnetic beads and washing away the remaining solvents and debris.  

There are two types: competitive and noncompetitive. In heterogenous competitive immunometric assay, the antibody is immobilized on a solid surface. An analyte consists of a mixture of antigens that compete for common binding site and one of the antigens is labeled for quantification.

Characteristics of enzymes used in Immunoassays

Ideally, the antigen-antibody immunocomplex should not affect the catalytic activity of the enzyme. The catalytic potential of enzymes can amplify the effect. The ideal features of the enzyme for immunoassays include purity, high specific activity, and stability at room temperature, high turnover number, simple detection and measurement of the enzyme activity, cheap and large-scale production.

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 which absorbs 410 nm and 650 nm light 
  • OPD (o-phenylenediamine dihydrochloride) substrate for HRP that produces a yellow-orange product which absorbs 492 nm 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 325 nm and emission at 420 nm. 
  • 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


Application of enzyme immunoassays

  • Clinical: enzyme immunoassay principles are used widely in:

    • Disease markers for heart disease measuring levels of CK-MB, prostate cancer detecting prostate-specific antigen;
    • Endocrine system for treatment of thyroid patients, detection of hormones, insulin assessing hypoglycemia;
    • Hypersensitivity reactions for Allergen study in asthma and other allergic reactions;
    • Cell viability assays for cytotoxic drug adverse effects in cancer patients;
    • Veterinary immunodiagnostics;
    • Drug abuse screening;
    • Vaccination studies for Viral markers; 
    • Epidemiology studies for the detection of infectious diseases;
    • Medicine industries such as sports anti-doping for prohibited recombinant human growth hormone (rhGH, rGH, hGH, GH); 
    • Rapid pregnancy test kits for detecting the pregnancy marker “human chorionic gonadotropin”;
    • Pharmaceutical industry for detection and/or quantitative measurement of Active Pharmaceutical Ingredients (APIs), drug discovery and analysis, therapeutic medication monitoring, conventional medicinal, bioequivalence and quality control of drugs.
  • Food, animal feed and beverages: The growing awareness regarding the foodborne microbes and chemicals, mycotoxins, bacterial toxins, pathogens, nutrient analysis in milk and baby foods, adverse effects of food additives (e.g. preservatives, contaminants, and adulterants) and food allergens among the general public initiated the application of immunoassay techniques by food research facilities and industries.
  • Water analysis for the detection of water pollutants and safety analysis;
  • Agriculture for the detection of endotoxins, pesticides and testing of seed and grain;
  • Environment for the detection of Industrial chemicals (e.g. Pesticides, Herbicides, Surfactants);
  • Basic research such as the study of biomolecular interactions and detection of biomolecules.


Current advances in the field of Enzyme Immunoassay

In the recent decades, enzyme immunoassay has been modified or complemented with other sensitive immunoassay techniques namely as Radioimmunoprecipitation Assay  and Optoelectronic Immunosensor–Based Assays. Furthermore, several detection systems with higher sensitivity are being designed based on the knowledge of optical physics, nanotechnology, and other fields such as Computer-Assisted Molecular Modeling in Immunoassay.

Compared to the other enzyme immunoassays (e.g. ELISA) magnetic bead CLIAs offer benefits as higher sensitivity, higher detection rate due to conjugated chemiluminescent label, faster performance, cost-benefit, concurrent detection-isolation function, magnetic beads size selection based on the analyte in question to have the highest yield of purification, an amplified signal and decreased matrix interferences and improved separation of the unbound reagents.

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