ELISA- Principle, Types, Uses, Advantages and Disadvantages

ELISA – Enzyme-Linked Immunosorbent Assays

Introduction

  • Immunoassays are bioanalytical methods, used to detect antigen-antibody specific reactions and provide qualitative /quantitative analysis in biological samples.
  • Researchers use enzyme-linked immunosorbent assays, commonly known as ELISA or EIA, as a type of immunoassay; furthermore, it is a modified version of classic radioimmunoassay, as it replaces radioisotopes with enzymes, which are considered safer than RIA.
  • For ELISA, researchers use a number of enzymes, which include alkaline phosphatase, horseradish peroxidase, and β-galactosidase.
  • Researchers commonly use ELISA in clinical diagnosis, food testing, drug discovery, and monitoring.

Principle

  • ELISA is based on antigen-antibody reactions, it represents the chemical interaction of antigen and antibody produced by immune cells i.e., leukocytes
  • ELISA allows selective quantitative/ qualitative analysis of antigens which also includes protein, peptides, hormones, nucleic acids, metabolites.
  • To detect molecule, enzyme labelled antibody or antigen is used.
  • Antigen is immobilised on a microtiter plate, made up of polyvinyl chloride, polypropylene and rigid polystyrene.
  • The primary antibody reacts with immobilised antigen; subsequently, it is later detected with enzyme-conjugated secondary antibody. Moreover, the presence of antigen is determined by the development of colour by using chromogenic substrates. For example, alkaline phosphatase produces p-nitrophenol (yellow colour) when p-nitrophenol phosphate is hydrolysed by the enzyme, and it is then detected at 405 nm.
  • Chemiluminescent substrates and fluorogenic substrates, on the other hand, can also be used for more sensitive detection; moreover, they offer distinct advantages in various applications.
  • Enzyme- substrates reaction completes within 30- 40 mins, the reaction is stopped by using sodium hydroxide, hydrochloric acid, etc.
  • A microtiter plate reader is used to detect, coloured and fluorescent products.

Types of ELISA

Researchers can use each type of ELISA qualitatively to detect the presence of antibodies and antigens. They can also determine the unknown concentrations of samples by using a standard curve with known concentrations of antibodies or antigens.

  • Direct ELISA was first developed in 1971, it set the base style for other types of ELISA with modifications. In this technique, antibody or antigen was immobilised on a microtiter plate, enzyme-conjugated antibodies were allowed to react followed by colour development with appropriate substrates which was measured.
  • These are common types of ELISA that are generally used:
    • Indirect ELISA
    • Sandwich ELISA
    • Competitive ELISA

Indirect ELISA

Indirect elisa

  • Add the sample containing primary antibody (Ab1) or serum to the microtiter well coated with antigen and allow it to react with the attached antigen in the well. [Fig – A & B]
  • Wash the free antibody (Ab1), and add an enzyme-conjugated secondary antibody to detect the bound antibody-antigen, which binds the primary antibody (Ab1).  [Fig C]
  • Wash the free secondary antibody and add the chromogenic substrate for an enzyme.
  • A researcher uses a specialised spectrophotometric plate to measure the amount of the coloured products formed by reading absorbance.

Uses

  • Indirect ELISA is suitable to detect the presence of serum antibodies (disease-associated antibodies), against endocrine diseases, human immunodeficiency virus (HIV), the causative agent of AIDS ( Acquired Immunodeficiency Syndromes).
  • For HIV antigen detection, the assay adsorbs core proteins and recombinant envelope of HIV as antigen immobilised to wells.
  • For individuals affected with HIV, serum antibodies will produce for viral protein and react with its epitopes.
  • Indirect ELISA can detect these serum antibodies within 6 weeks of infection.

Sandwich ELISA

Sandwich ELISA

  • Researchers use Sandwich ELISA to detect or measure antigens.
  • In this technique, researchers immobilise primary antibody on microtiter well plates [A]; subsequently, when they add a sample containing antigens, it reacts with the immobilised antibody [B].
  • After washing the well, we add the enzyme conjugated secondary antibody specific for different epitopes and permit it to react with the bound antigen. [C]
  • After washing any free secondary antibody, the technician adds substrate and measures the coloured reaction product.

Uses

  • Used to detect macromolecules such as toxins or proteins.
  • For instances, monoclonal antibodies in sandwich ELISA system can detect Ciguatoxins, causative toxins of ciguatera seafood poisoning, produced by marine dinoflagellate Gambierdiscus toxicus.
  • Sandwich ELISA can also detect marine biotoxins e.g., palytoxins.

Competitive ELISA

Competitive ELISA

  • Competitive ELISA can measure the amount of antigen.
  • In this technique, the primary antibody is first incubated in a solution containing the antigen. [A]
  • We add the Antigen-Antibody mixture to the antigen immobilised to the microtiter well. [B]
  • More antigens are present in the sample, less free antibodies will be present to bind to the antigen-coated well.
  • Researchers add an enzyme-conjugated secondary antibody specific for the isotype of the primary antibody to determine the amount of primary antibody bound to the antigen-coated well, and they measure the coloured reaction products. [C & D]
  • In a competitive assay, absorbance is lower when, in fact, the concentration of antigen in the original sample is higher.

Uses

  • Competitive ELISA can detect both macromolecules and low molecular weight hapten.

Advantage of ELISA

  • Simple Procedure
  • High Specificity and sensitivity – Due to antibody-antigen reactivity
  • High Efficiency- You do not need complicated pre-treatment, and you can perform analysis simultaneously.
  • Considered safe and Eco- Friendly
  • Cost-Effective Assays

Disadvantage of ELISA

  • Antibody Preparations is an expensive and laborious process
  • High possibilities of false-positive and negative.
  • Antibody Instability
  • Antibodies require refrigerated transport and storage.

References and Sources

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