Principle of immunoassays

The basic principle of these assays is the specificity of the antibody-antigen reaction. There are several different formats of immunoassays (see below under “Types of immunoassays”) but all of them involve a specific antibody binding to its antigen, and a label that is used to detect the antigen-antibody complex.

The label can be linked to the primary antibody that binds to the antigen, to a secondary antibody binding to the primary antibody, or to a portion of antigen that is added to the sample and competes with the antigen to be measured. To separate the bound from the free fraction, at least one washing step is needed. Actually, as immunoassays usually involve multiple washing, dispensing and incubation steps, assay workstations are often used to automate the process.

Types of immunoassays

Immunoassays by signal

A useful way to classify immunoassays is by the type of signal that is measured, as this has an impact on its sensitivity and the instrumentation needed to measure them. The most usual types of immunoassays are:

  • ELISA (Enzyme-Linked Immunosorbent Assay) if the signal is provided by an enzyme that produces a coloured substrate that is measured using absorbance. They are usually measured using an ELISA reader or in an ELISA workstation that automates the complete assay. They are the most common type of immunoassay, but ELISA has limited sensitivity, that can be improved by moving to more sensitive detection methods.
  • FIA (Fluorescent ImmunoAssay) if the signal is provided by fluorescent dyes (e.g. FITC); in this case the measurement is performed by using a microplate fluorometer.
  • CLIA (ChemiLuminescent ImmunoAssay) if the signal is provided by an enzyme that catalyses a reaction which produces light. They are measured using a microplate luminometer. Luminescence is one of the detection methods that provides the highest sensitivity.
  • RIAs (RadioImmunoAssay) if the signal is provided by radiation coming from a radioisotope, such as 125I. As they are often gamma-emitting isotopes, they are usually measured using a gamma counter. Radiation is another of the detection methods that provides the highest sensitivity.
  • Newer detection technologies have also been applied to immunoassays, such as TRF and Alpha technology.

Immunoassays by Format

Several different immunoassay formats exist, depending on what antigen-antibody complexes are formed and where is the signal to be quantified. As terminology comes from the most usual immunoassay, ELISA, they are often called by the name of the respective ELISA formats:

  • Direct ELISA: the labelled antibody binds directly the antigen of interest.
  • Indirect ELISA: the labelled antibody binds the antibody of interest, that is bound to an immobilized antigen.
  • Sandwich ELISA: the antigen of interest is captured between an immobilized antibody and the labelled antibody.
  • Competitive ELISA: the sample antigen competes with an inhibitor antigen, binding to a limited amount of labelled antibody.

You can find more information about immunoassay formats in our page ELISA formats.

Applications of immunoassays

Immunoassays are widely used in research, drug discovery and diagnostics for highly specific and cost-efficient detection of analytes including, but not limited to:

  • Tumour Markers, e.g. AFP, CEA, hCG, PSA…
  • Cardiac Markers, e.g. CK-MB, CRP, Digoxin, Myoglobin…
  • Cell-based Assays, e.g. cell cytotoxicity…
  • Allergy, e.g. histamines, egg, milk, allmonds…
  • Growth Deficiency, e.g. hGH
  • Enzyme activity
  • Hormone and Steroid Screening, e.g. T4, fT3, TSH…
  • Drug Abuse Screening, e.g. amphetamines, cocaine, LSD…
  • Immunological Screening
  • Infectious Diseases, e.g. Chlamydia, CMV, Hepatitis, Rubella…
  • Veterinary, e.g. bacterial infection, fertility, drugs, BSE…
  • Food and Beverages, e.g. pathogens, toxins…
  • Water Analysis, e.g. bacterial contamination, toxins, heavy metals…
  • Agriculture, e.g. endotoxins, pesticides…
  • Environment, e.g. industrial chemicals, pesticides, surfactants…

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Instruments recommended for Immunoassays