Immunoassays are analytical techniques that are widely used to detect and quantitate the concentration of a protein, metabolite, or drug in a biological sample. They are performed in essentially every clinical laboratory in the world today. Results are used by doctors to screen and diagnose diseases, monitor the progress of therapy, and predict those who will develop medical problems in the future. Immunoassays are also the mainstays in research, for example, understanding the pathophysiology of medical disorders in order to develop novel drugs. In order to meet specific testing needs, immunoassays have been developed in a variety of formats. Most of them depend on the unique features of the reaction between an antigen and its complementary antibody to achieve specificity for the analyte of interest. However, other antigen‐binding materials are increasingly being used.

Key Concepts

  • Antibodies are raised in non‐human species targeted to specific epitope of the intended target (antigen).
  • Some assays are subjected to interferences due to the presence of unusual antibodies in the sample (heterophile and human anti‐animal antibodies).
  • The use of chimeric and humanised antibodies improves the performance of immunoassays over those antibodies that are 100% animal.
  • Heterogeneous immunoassays require a physical separation between antibody‐bound and free analytes.
  • Homogeneous immunoassays do not require a physical separation step.
  • Antigens present in excess concentrations can produce falsely low results in both immunoprecipitation (prozone) and immunometric (hook effect) assays.
  • Point‐of‐care devices make use of immunochromatographic assays.
  • There are a variety of detectors used in immunoassays such as enzymes, fluorophores, luminescent tags, electrochemical reactions and immunosensors.
  • Multiplexing immunoassays can be accomplished through microbead or microchip arrays.

Keywords: antigen antibody reaction; affinity; avidity; heterogeneous and homogeneous immunoassays; enzyme; fluorescence; luminescence

Figure 1. Schematic representation of the immunoglobulin G molecule: red, heavy chain; orange, light chain; dotted circle, paratope region.
Figure 2. Antibody structure. (a). Mouse antibody (all in blue). (b). Human antibody (all in brown). (c). Chimeric antibody. The Fab portion is derived from the mouse (in blue), whereas the Fc portion is from the human (in brown). (d). Humanised antibody. Parts of the Fab portion are derived from the mouse (blue stripes) but retain recognition against the antigen. The Fc portion is from the human (brown). Reproduced with permission from Bishop M, Fody EP, Schoeff LE, Clinical Chemistry Principles, Techniques, and Correlations, 7th ed., Lippincot Wolters Kluwer Healtht Williams and Wilkins, 2017: Philadelphia, PA. © Wolters Kluwer Health.
Figure 3. Diagrammatic representation of the typical relationship between the amount of immunocomplex formed when a variable amount of protein antigen reacts with a fixed amount of antibody (—). This is known as the Heidelberger–Kendall curve. The left‐hand sector (‐‐‐) denotes the type of calibration curve found in an immunometric excess‐reagent assay. The right‐hand sector (…) denotes the type of calibration curve found in a competitive limited‐reagent assay.
Figure 4. Schematic representation of competitive limited‐reagent (a) and immunometric excess‐reagent (b) heterogeneous immunoassays.
Figure 5. Schematic representation of a homogeneous immunoassay in which, when present, the sample antigen binds to the antibody, thereby allowing access of a substrate to the active site of an enzyme labelled with the antigen. In the absence of sample antigen, the antibody binds to the antigen attached to the enzyme, inhibiting access to the substrate, thereby effectively modulating the activity. The split box substrate denotes that the enzyme is acting to split the substrate.
Figure 6. Schematic representation of an immunochromatographic device.
Figure 7. Drug‐of‐abuse immunoassay testing on a chemistry analyser versus a microchip array. (a): A typical chemistry analyser has a limited number of agent storage containers (‘wedges’) loaded onto the instrument. In order to detect as many drugs as possible, antibodies are generalised to specific classes of compounds (e.g. drug classes such as amphetamines, opiates and benzodiazepines). (b): A microarray chip does not make use of liquid antibody reagents. Instead, they are preloaded onto the chip. In this example, because there are 96 locations on the chip, each can be designated as a single assay (codeine, morphine, oxycodone, individual opiate drugs). This approach enables use of more antibodies that are specific to the individual drugs rather than the drug class, thereby improving the assay's specificity.


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Further Reading

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Masseyeff RF , Albert WH and Staines NA (eds) (1993) Methods of Immunological Analysis, vol. 2. Samples and Reagents, vol. VCH.: Weinheim.

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Wu, Alan(May 2017) Immunoassay. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001135.pub2]