A Brief Overview of Immunoassay [Principles of Immunoassay, Types & Applications]

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A Brief Overview of Immunoassay [Principles of Immunoassay, Types & Applications]

Biochemists, pharmacists, microbiologists, and researchers in fields of medicine, biotech, genetics, and nanotechnology are familiar with this terminology. These personals are used to perform immunoassay as routine work in laboratories, pathological labs, research centers, and pharmaceutical companies for medical and research purposes. Currently, the immunoassay test for covid 19 is a burning issue due to the pandemic of coronavirus. Immunoassay is the standard procedure and method used in medicines for multiple beneficial effects. This article is a complete package to understand 

  • Immunoassay 
  • Immunoassay principles
  • Immunoassay types
  • Applications of immunoassay

What is Immunoassay?

Immunoassay can be defined as a biomedical procedure or test used to detect the presence or measure the concentration of a molecule ( either small or macromolecule) in a solution (body fluid or blood) by means of using an antibody or antigen. Immunoassays are performed to locate the presence of an analyte ( antigen or antibody). Immunoassays are frequently performed for medical and research purpose  to detect analytes in biological liquids such as  be urine or serum 

Immunoassay Principle:

The immunoassay principle helps to understand that how to do immunoassay test works. 

The immunoassay principle depends on the ability of antibodies to detect, recognize and bind a specific macromolecule ( antigen) or in a mixture of complex macromolecules. The area on antigen where the antibody binds is known as an epitope. Here analyte is an antigen

In some situations, Immunoassay is performed to detect the presence of specific antibodies in a solution ( it may be blood or body fluid). This can be done by addition of antigen and allow it to react with that antibody to form an antigen-antibody complex. Here analyte is an antibody  

In all types of immunoassay, the major key feature is to develop a measurable signal in response to the binding. 

Types of Immunoassay:

There are five types of immunoassay:

  • Enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA):

 In this type of immunoassay, an antibody is attached to an enzyme. It is then allowed to react with antigen during incubation. Unbound antibody is washed away. a substrate is added to the solution and the activity of the bound antibody-enzyme attached to the target antigen is observed. A chemical reaction of the substrate is catalyzed by the enzyme to produce a noticeable color change.

Enzyme Immunoassay
  • Radioimmunoassay (RIA): 

It is the oldest type of immunoassay in which a radioisotope is linked with an antigen of interest. It is then allowed to bound with a complementary antibody. Proceeding forward, a sample with antigen to be measured is added that exhibits competition with radioactive antigen and competes at its binding site to replace it. The radioactivity of the sample is detected after washing of unbound antigens. This possess may cause health hazards and not safe at all.

  • Fluro-Immunoassay (FIA): 

This type of immunoassay required the labeling of antigens with fluorescent probes. These antibodies then incubated with antigen to form an antigen-antibody complex. This complex is isolated and fluorescent intensity is measured. 

  • Chemiluminescent Assay (CLIA): 

The immunoassay principle of  Chemiluminesce is similar to fluorescence with the difference that here luminescence occurs due to chemical reaction. Whereas in fluorescence, luminescence takes place with help of certain frequencies of light. 

  • Counting Immunoassay (CIA): 

in this type of immunoassay, polystyrene beads are coated with many antibodies complementary to the target antigen. These beads bind to multiple antigens during incubation, while a few beads remain unbound. The whole solution is allowed to pass through a cell counter that only counts unbound beads. There is an inverse relationship between the number of unbound beads to the amount of antigen.

Applications of Immunoassay:

Immunoassay is the bioanalytical method with various applications in different fields such as the pharmaceutical industry, food industries, clinical diagnosis, and research work. We discuss its applications in specific field briefly  with examples 

Immunoassay: In the Pharmaceutical Analysis:

In the pharmaceutical analysis

In pharmaceutical analysis, all types of immunoassay are extensively utilized to detect the analyte or quantify it. Pharmaceutical analysis usually covers the area of drug discovery and development, clinical pharmacokinetics, and bioequivalence. Immunoassay in the pharmaceutical analysis is performed for a variety of purposes mentioned as follows:

  • In the measurement of low molecular weight or low concentration drugs 
  • To detect the biomolecule, metabolite, or biomarker  of pharmaceutical interest
  • To determine the unknown concentration by the standard calibration curve.
  • Radio-immunoassay is successfully used to determine a limitless number of pharmaceutically important compounds in biological fluid.
  • In new drug discovery and development, immunoassay is utilized to carry out an accurate analysis.
  • Enzyme-linked immunoassay is used to check enzyme activity and its binding with the target antigen

In The Food Industry:

Immunoassay In The Food Industry

Immunoassay is being performed in the food industry to evaluate the raw material and final composition of food products. The objective of immunoassay in the food industry is to detect any contamination that may cause life hazards before the marketing and consumption of the food products. The significance of immunoassay can be highlighted as follows:

  • Immunoassay is performed to test foodborne pathogen ( specifically E.coli and Listeria) by utilizing polyclonal antibodies
  • Enzyme-linked immunoassay or ELISA is used for mycotoxin testing 
  • Immunoassay is performed to detect food allergens as food allergies may be fatal and cause serious consequences.

In Analytical Toxicology:

Drug immunoassay has been successfully utilized in clinical research and to evaluate toxic concentration. Toxicology studies require the quantification of the simple to determine the concentration that causes acute or chronic toxicity. It is somehow linked with clinical pharmacokinetics and drug development. Immunoassay is used to develop the toxicity profile for a new drug candidate. 

Immunoassay Test for Covid-19:

Rapid covid-19 strip Test

By the end of 2019, the Novel coronavirus has been cause enough destruction and challenge the health care system in terms of its diagnosis and treatment. So efforts have been made to diagnose the presence of a covid virus on the basis of immunoassay. Four types of Enzyme-linked immunosorbent assay are approved by FDA. The study reveals that specificity levels against recombinant structural protein from SARS-CoV-2 are 91% and 73% respectively. Microsphere immunoassay is more specific and sensitive than lateral immunoassay and can be utilized for the detection of a strain of coronavirus. 


Immunoassay is the biochemical and bioanalytical method that is utilized as a major analytical method due to its sensitivity, accuracy, and specificity. Its basic principle includes the antigen-antibody reaction and detects the presence or concentration of the desired analyte. Immunoassay for covid19 is also under study and research. Different types of immunoassay are used according to the type of analysis and nature of the analyte. Immunoassay owns major significance in pharmaceutical analysis and toxicological studies. 

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