Image of enzyme linked immunosorbent assay.
Image of enzyme linked immunosorbent assay


In this article, we will discuss about the enzyme linked immunosorbent assay. It is a biochemical technique use to detect the presence of antigen, antibodies, proteins and other biological molecules in a sample. The principle of ELISA is based the interaction of antigens and antibodies. It used in detection of diseases and pathogens, concentration of antigen and antibodies and several other biological applications. We will also discuss about the procedure of enzyme linked immunosorbent assay technique. We will also provide related references to understand the concept deeply.


It is a biomolecular technique that utilizes the specificity of an antibody, as well as the sensitivity of enzyme assays, to detect and quantify molecules such as hormones, peptides, antibodies, and proteins. The principle of ELISA based on the specific interaction of antigen and antibodies. Enzyme immunoassays (EIAs) use the catalytic properties of enzymes to detect and quantify immunologic reactions. It is a heterogeneous EIA technique use in clinical analyses. It helps to examine the presence of antibodies in the body, in case of certain infectious diseases. ELISA yields quantitative results and separation of non-specific and specific interactions that take place through serial binding to solid surfaces, which is normally a polystyrene multi-well plate.


The principle of an ELISA relies on the specific binding between an antibody and antigen. This specificity comes from the unique three-dimensional structure of the antibody paratope and the antigen epitope. These two regions fit like a lock and key, via non-covalent, charge-based, and/ or hydrophobic interactions. When an ELISA is used to detect antigen presence, it provides a better understanding of current infection as the antigen is only present in an active infection. In order to increase the sensitivity and precision of the assay, the plate must be coated with antibodies with high affinity.

Image of principle of enzyme linked immunosorbent assay.
Image of principle of enzyme linked immunosorbent assay


ELISAs are performed in polystyrene plates, typically 96-well plates coated to bind protein strongly. Depending on the ELISA type, testing requires a primary and/or secondary detection antibody, analyte/antigen, coating antibody/antigen, buffer, wash, and substrate/chromogen. The primary detection antibody is a specific antibody that only binds to the protein of interest. In contrast, a secondary detection antibody is a second enzyme-conjugated antibody that binds to a primary antibody that is not


  1. Prepared the standard solution in which we have to detect the antigen, antibodies or other molecules.
  2. Diluted the sample with dilution buffer.
  3. An antibody attached to a polystyrene plate which is a solid surface and attracted or has an affinity towards bacteria, other antibodies and hormones.
  4. A microtiter coated with antigen, filled with this antigen-antibody mixture after which free antibodies removed by washing.
  5. A second antibody specific to primary antibody added which usually conjugated with an enzyme.
  6. Free enzyme-linked secondary antibodies removed by washing the plate.
  7. Finally, the substrate added. The substrate converted by the enzyme to form a colored product, which can measured by spectrophotometry.
  8. After the results measured, a standard curve from the serial dilutions data plotted with a concentration on the x-axis using a log scale and absorbance on the y-axis using a linear scale.
Procedure of enzyme linked immunosorbent assay
Procedure of enzyme linked immunosorbent assay


  1. The presence of antibodies and antigens in a sample can determine.
  2. It used in the food industry to detect any food allergens present.
  3. To determine the concentration of serum antibody in a virus test.
  4. During a disease outbreak, to evaluate the spread of the disease, e.g. during recent COVID-19 outbreak, rapid testing kits being used to determine presence of antibodies in the blood sample.
  5. Enzyme-linked immunosorbent assays are applied in many diagnostic tests.
  6. Detect and Measure the Presence of Antibodies in the Blood. Detect and Estimate the Levels of Tumor Markers.
  7. Detect and Estimate Hormone Levels.
  8. Tracking Disease Outbreaks.
  9. Detecting Past Exposures.
  10. Screening Donated Blood for Possible Viral Contaminants.
  11. Detecting Drug Abuse.


Results fetched from ELISA gives an accurate diagnosis of a particular disease since two antibodies used. Can be carried out for complex samples as the antigen is not required to get purified to detect. It is highly responsive since direct and indirect analysis methods can carried out. It is a rapid test, yields results quickly. Possible detection for ELISA ranges from the quantitative, semi-quantitative, standard curve, qualitative, calibration curve models etc. Easier to perform and uncomplicated process as compared to other assays which require the presence of radioactive materials. ELISAs can used in many settings, including rapid antibody screening tests for Human immunodeficiency virus (HIV),
detection of other viruses, bacteria, fungi, autoimmune diseases, food allergens, blood typing, presence of the pregnancy hormone hCG, laboratory and clinical research, forensic toxicology, and many other diagnostic settings.


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