Immunoassays

Immunoassays are plate-based techniques that can detect and quantify many types of molecules through antibody-antigen reactions. An immunoassay typically involves an analyte, a targeted antibody, and labels. Classification of immunoassays is based on the type of label utilized, which includes enzymes (ELISA), light-emitting molecules/tracers (e.g., chemiluminescence and fluorescence immunoassays), and radioactive isotopes (radioimmunoassays). These specialized immunoassays are relatively sensitive, specific, inexpensive, and rapid, and are widely used in a clinical setting. Immunoassays are used in the diagnosis of infectious diseases, identification of tumor markers, allergy testing, and monitoring drug levels.

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Editorial responsibility: Stanley Oiseth, Lindsay Jones, Evelin Maza

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Overview

Definition

Immunoassays are plate-based assay techniques designed for detecting and quantifying peptides, proteins, antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins, and hormones Hormones Hormones are messenger molecules that are synthesized in one part of the body and move through the bloodstream to exert specific regulatory effects on another part of the body. Hormones play critical roles in coordinating cellular activities throughout the body in response to the constant changes in both the internal and external environments. Hormones: Overview. The most crucial element of the detection strategy is a highly specific antibody-antigen reaction.

Components

  • Analyte: the molecule of interest (antigen)
  • Antibody: carefully selected for the specific analyte
  • Labels:
    • Molecules that have the potential to conjugate with the antibody-antigen complex
    • Allow for detection and quantification

Types of immunoassays

The type of label defines the immunoassay being performed:

  • Enzymes: ELISA
  • Specialized molecules/tracers (along with enzymes Enzymes Enzymes are complex protein biocatalysts that accelerate chemical reactions without being consumed by them. Due to the body's constant metabolic needs, the absence of enzymes would make life unsustainable, as reactions would occur too slowly without these molecules. Basics of Enzymes) that have the property of light emission:
    • Chemiluminescence
    • Fluorescence immunoassay (FIA)
  • Radioactive isotopes: radioimmunoassay

Procedure

General process

  • A sample/analyte is added to a plate.
  • An antibody and a label are added.
  • An antibody-antigen complex forms.
  • A washing step is performed in some assays to remove unbound antigen/ antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins.
  • A substrate is added, which reacts with the label and results in:
    • Color change (ELISA)
    • Luminescence (chemiluminescence)
    • Fluorescence (FIA)
    • Radiation emission (radioimmunoassay)
  • Changes/signals are detected to determine the presence/amount of antigen in a sample. Detection methods may include:
    • Spectrometry (color change)
    • Luminometry (light emission)
    • Gamma counter (radiation)

Variants of ELISA

There are 4 major types of ELISA, which are variations of the general immunoassay process:

  • Direct ELISA:
    • The plate is coated with an antigen.
    • Incubation with a specific, labeled antibody
  • Indirect ELISA:
    • The plate is coated with an antigen.
    • Incubation with a primary, unlabeled antibody (specific to the antigen)
    • Incubation again with a secondary, labeled antibody (specific to the primary antibody)
  • Sandwich ELISA (analyte is “sandwiched” between 2 layers of antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins):
    • An antibody-coated plate is used.
    • The analyte antigen is added to the plate and incubated.
    • Incubated again with a labeled antibody
  • Competitive ELISA:
    • An antibody-coated plate is used (an antigen-coated plate is used if testing for a specific antibody).
    • Sample target antigen (or antibody) is added.
    • Labeled target antigen (or antibody) is added and then washed.
    • Note: Unlike other ELISA methods, less color/absence of color indicates a positive result.

Uses

Immunoassays have a wide range of clinical applications. The examples listed below are not exhaustive.

Infectious diseases

Immunoassays can be used to directly identify microorganisms (based on antigens or toxins) or indirectly assess for antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins to the infectious agent. Some examples include:

  • Microorganism detection:
    • Legionella Legionella Legionella is a facultative intracellular, gram-negative bacilli. Legionella does not grow on common culture media because it requires certain supplementation (cysteine and iron). Legionella pneumophila (L. pneumophila) accounts for the majority of human infections. Legionella/Legionellosis pneumophila
    • Escherichia coli Escherichia coli The gram-negative bacterium Escherichia coli is a key component of the human gut microbiota. Most strains of E. coli are avirulent, but occasionally they escape the GI tract, infecting the urinary tract and other sites. Less common strains of E. coli are able to cause disease within the GI tract, most commonly presenting as abdominal pain and diarrhea. Escherichia coli (toxin)
    • Clostridioides difficile (toxin)
    • Hepatitis B Hepatitis B Hepatitis B virus (HBV) is a partially double-stranded DNA virus, which belongs to the Orthohepadnavirus genus and the Hepadnaviridae family. Most individuals with acute HBV infection are asymptomatic or have mild, self-limiting symptoms. Chronic infection can be asymptomatic or create hepatic inflammation, leading to liver cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B Virus
  • Antibody detection:
    • HIV
    • West Nile virus West Nile Virus West Nile virus is an enveloped, positive-sense, single-stranded RNA virus of the genus Flavivirus. Birds are the primary hosts and the disease is most often transmitted by Culex mosquitoes. Most people infected with West Nile virus are asymptomatic. Some patients develop West Nile fever (a self-limited, febrile illness) and a very small proportion of patients develop West Nile neuroinvasive disease. West Nile Virus
    • Hepatitis C Hepatitis C Hepatitis C is an infection of the liver caused by the hepatitis C virus (HCV). The infection can be transmitted through infectious blood or body fluids and may be transmitted during childbirth or through IV drug use or sexual intercourse. Hepatitis C virus can cause both acute and chronic hepatitis, ranging from a mild to a serious, lifelong illness including liver cirrhosis and hepatocellular carcinoma (HCC). Hepatitis C Virus
    • Borrelia Borrelia Borrelia are gram-negative microaerophilic spirochetes. Owing to their small size, they are not easily seen on Gram stain but can be visualized using dark-field microscopy, Giemsa, or Wright stain. Spirochetes are motile and move in a characteristic spinning fashion due to axial filaments in the periplasmic space. Borrelia burgdorferi 
    • Treponema Treponema Treponema is a gram-negative, microaerophilic spirochete. Owing to its very thin structure, it is not easily seen on Gram stain, but can be visualized using dark-field microscopy. This spirochete contains endoflagella, which allow for a characteristic corkscrew movement. Treponema pallidum

Cancer detection and monitoring

Immunoassays can be utilized to detect tumor markers. Examples include:

  • PSA
  • Cancer antigen-125
  • Alpha-fetoprotein (AFP)
  • Carcinoembryonic antigen (CEA)

Allergy testing

Immunoassays can be used to detect IgE antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins to specific allergens:

  • Positive result:
    • Indicates sensitization to that allergen
    • Does not necessarily indicate whether that individual will have a clinical response upon exposure to that antigen
  • Negative result:
    • Suggests no allergy to the antigen
    • Does not completely exclude allergy to the antigen (particularly, if there is a suggestive clinical history)

Medications

Therapeutic drug monitoring is an important application of immunoassays. Examples include monitoring the drug levels of:

  • Digoxin
  • Theophylline
  • Immunosuppressants Immunosuppressants Immunosuppressants are a class of drugs widely used in the management of autoimmune conditions and organ transplant rejection. The general effect is dampening of the immune response. Immunosuppressants (e.g., mycophenolic acid, cyclosporine)
  • Antibiotics (e.g., amikacin, vancomycin, gentamicin)

Other diagnostic uses

Additional laboratory tests where immunoassays may be utilized include:

  • High-sensitivity troponin I
  • Thyroid function:
    • Thyroid-stimulating hormone (TSH)
    • Free thyroxine (T4) and triiodothyroonine (T3)
  • Nutritional:
    • Folate Folate Folate and vitamin B12 are 2 of the most clinically important water-soluble vitamins. Deficiencies can present with megaloblastic anemia, GI symptoms, neuropsychiatric symptoms, and adverse pregnancy complications, including neural tube defects. Folate and Vitamin B12
    • Vitamin B12 Vitamin B12 Folate and vitamin B12 are 2 of the most clinically important water-soluble vitamins. Deficiencies can present with megaloblastic anemia, GI symptoms, neuropsychiatric symptoms, and adverse pregnancy complications, including neural tube defects. Folate and Vitamin B12
    • Ferritin
    • Vitamin D

Advantages and Errors

Advantages

Advantages depend on the type of immunoassay used; however, generally, they are:

  • Inexpensive
  • Sensitive
  • Specific
  • Relatively rapid and convenient

Sources of error

  • Cross-reactivity (reagent antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins bind to molecules that are similar to the analyte → false positive/falsely elevated results)
  • Autoantibodies (endogenous antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins, instead of reagent antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins) bind to the analyte → false negative/falsely low results)
  • Antireagent antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins (endogenous antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins bind to reagent antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins → false positive/falsely elevated results)

References

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  2. Engvall, E. (2010). The ELISA, enzyme-linked immunosorbent assay. Clin Chem. 56(2), 319-320. [PubMed]
  3. Shah, K., Maghsoudlou, P. (2016). Enzyme-linked immunosorbent assay (ELISA): the basics. Br J Hosp Med (Lond). 77(7), C98-C101. [PubMed]
  4. Konstantinou, G.N. (2017). Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol. 1592, 79-94. [PubMed]
  5. Kohl, T.O., Ascoli, C.A. (2017). Direct competitive enzyme-linked immunosorbent assay (ELISA). Cold Spring Harb Protoc. 2017(7):pdb.prot093740. [PubMed]
  6. Kohl, T.O., Ascoli, C.A. (2017). Indirect immunometric ELISA. Cold Spring Harb Protoc. May 01;2017(5) [PubMed]
  7. Kohl, T.O., Ascoli, C.A. (2017). Immunometric double-antibody sandwich enzyme-linked immunosorbent assay. Cold Spring Harb Protoc. Jun 01;2017(6):pdb.prot093724. [PubMed]
  8. Kowal, K., DuBuske, L. (2021). Overview of in vitro allergy tests. In Feldweg, A.M. (Ed.), UpToDate. Retrieved December 28, 2021, from https://www.uptodate.com/contents/overview-of-in-vitro-allergy-tests
  9. Alhajj, M., Farhana, A. (2021). Enzyme linked immunosorbent assay. [online] StatPearls. Retrieved December 28, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK555922/
  10. Gleichmann, N. (2020). Immunoassays: A guide. Technology Networks. Retrieved December 28, 2021, from https://www.technologynetworks.com/diagnostics/articles/immunoassays-a-guide-338790
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