Enzyme-Linked Immunosorbent Assay (ELISA)

Definition of ELISA

The enzyme-linked immunosorbent assay is a commonly used analytical biochemistry assay. It was first described by Engvall and Perlmann in 1971. A solid-phase sort of enzyme is used as an immunosorbent which is used to detect the presence of a ligand that is commonly a protein, during a liquid sample using antibodies directed against the protein to be measured. This is why it is known as immunosorbent assay.  ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, also as a top-quality control sign for various industries.

To the surface antigens that are to be tested are attached. An antibody that is identical in nature is attached. This antibody is linked to an enzyme then any unbound antibodies are removed. within the final step, a substance containing the enzyme's substrate is added. If there was binding the next reaction produces a detectable signal, most ordinarily a colour change. The enzyme used in ELISA is horseradish peroxidase. Acetylcholinesterase and catalase are also the enzymes used in the ELISA test.

Materials for ELISA

Performing an enzyme-linked immuno sorbent assay ELISA involves a minimum of one antibody with specificity for a specific antigen. On solid support, the sample that has an unknown amount of antigen is attached. It is usually a polystyrene microtiter plate. After the antigen is immobilized, the detection antibody is added, forming a posh with the antigen. The detection antibody is often covalently linked to an enzyme or can itself be detected by a secondary antibody that's linked to an enzyme through bioconjugation. Between each step, the plate is usually washed with a light detergent solution to get rid of any proteins or antibodies that are non-specifically bound. After the ultimate wash step, the plate is developed by adding an enzymatic substrate to supply a clear signal, which indicates the number of antigens within the sample.

Antigen-antibody Binding

Immunosorbent meaning is that it is the technique by which antibodies are made. ELISA can perform other sorts of ligand binding assays also. Horseradish peroxidase is the enzyme used in ELISA or the enzyme-linked immunosorbent assay. The technique essentially requires any ligating reagent which will be immobilized on the solid phase alongside a detection reagent that will bind specifically and use an enzyme to get a sign which will be properly quantified. In between the washes, only the ligand, and its specific binding counterparts remain specifically bound or are immunosorbent by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where an equivalent reaction well is often reused after washing, the ELISA plates have the reaction products immunosorbent on the solid phase, which is a component of the plate, they are not easily reusable.


ELISA enzyme immunoassay involves the detection of an analyte that is the precise substance whose presence is being quantitatively or qualitatively analyzed during a liquid sample by a way that continues to use liquid reagents during the analysis. It is a controlled sequence of biochemical reactions which will generate a sign which may be easily quantified and interpreted as a measure of the quantity of analyte within the sample) that stays liquid and remains inside a reaction chamber or well needed to stay the reactants contained. Catalase is also an enzyme used in ELISA. 

To a stationary solid phase, a liquid-solid sample is added which is present with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated, and washed, followed by some optical change that can be colour developed by the merchandise of an enzymatic reaction within the final liquid within the well from which the number of the analyte is measured. The quantitative reading is typically supported detection of intensity of transmitted light by spectrophotometry, which involves quantitation of transmission of some specific wavelength of sunshine through the liquid as well because of the transparent bottom of the well within the multiple-well plate format. On the amplification of the signal during the analytic reactions, the sensitivity of the detection is dependent.  Since enzyme reactions are alright known amplification processes, the signal is generated by enzymes that are linked to the detection reagents in fixed proportions to permit accurate quantification, and thus the name enzyme-linked.

The analyte is also called the ligand because it helps in specifically binding or ligating to a detection reagent. This is the reason that Enzyme-linked immunosorbent assay falls under the larger category of ligand binding assays. The ligand-specific binding reagent is immobilized, which means it is usually coated and dried onto the transparent bottom and sometimes also sidewall of a well. The specificity of antigen-antibody type reaction is employed because for an antibody, it is easy to boost it,  specifically against an antigen in bulk as a reagent. Alternatively, if the analyte itself is an antibody, its target antigen is often used because of the binding reagent. 

Types of ELISA Assay

For particular molecules that use the method of matching antibodies, there are multiple enzyme-linked immunosorbent assay tests. The main types are described here: 

  • Direct ELISA: A buffered solution of the antigen to be tested for is added to every well of a microtiter plate, where it is given time to stick to the plastic through charge interactions. A solution of non-reacting protein, like bovine albumin or casein, is added to every well so as to hide any plastic surface within the well which remains uncoated by the antigen. The primary antibody with an attached enzyme is added, which binds specifically to the test antigen coating the well. A substrate for this enzyme is then added. The colour of the substrate is changed when it reacts with the enzyme. The higher the concentration of the first antibody present within the serum, the stronger the colour change. Often, a spectrometer is employed to offer quantitative values for colour strength.

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  • Sandwich ELISA: An antibody that has the ability to catch, is coated on the plate. Then the addition of the sample takes place. The antigen then captures the antibody. A detecting antibody is also added that helps in binding to the antigen. Then we add a secondary antibody which then binds to the detecting antibody. This is done so that the substrate is converted into a detectable form with the help of the enzyme. 

A "Sandwich" ELISA is Employed to Detect Sample Antigen. The Steps are: 

  • A surface is ready to which a known quantity of capture antibody is bound.

  • On the surface, the nonspecific binding sites are blocked.

  • The antigen-containing sample is applied to the plate and captured by an antibody.

  • The plate is washed to get rid of the unbound antigen.

  • A specific antibody is added and binds to the antigen This primary antibody could even be within the serum of a donor to be tested for reactivity towards the antigen.

  • The plate is washed to get rid of the unbound antibody-enzyme conjugates.

  • The chemical is added because it will be converted to an electrochemical signal. 

  • The absorbance or fluorescence or electrochemical signal of the plate wells is measured to work out the presence and quantity of antigen.

FAQs (Frequently Asked Questions)

1. Explain the Steps of Competitive ELISA.

Answer: The steps are: 

  • The unlabeled antibody is incubated within the presence of its antigen (sample).

  • These bound antibody/antigen complexes are then added to an antigen-coated well.

  • The plate is washed, so unbound antibodies are removed. (The more antigen within the sample, the more Ag-Ab complexes are formed then there are fewer unbound antibodies available to bind to the antigen within the well, hence competition. 

  • The secondary antibody, specific to the first antibody, is added. This second antibody is coupled to the enzyme.

  • A substrate is added, and the remaining enzymes elicit a chromogenic or fluorescent signal.

  • The reaction is stopped to stop the eventual saturation of the signal.

2. What are Antigens?

Answer: They are the molecules that when introduced into the body produce a chemical response. The chemical reaction or response is because of the presence of antibodies that are produced when the antigens or foreign molecules enter the human body. They are generally large molecules. They can be bacteria, fungi and viruses. But all microorganisms are not antigens. Certain pollen and fruits and vegetable pieces can also act as an antigen. The antigens are responsible for the immune response in our body.