Immunoassays

Overview

Definition

Immunoassays are plate-based assay techniques designed for detecting and quantifying peptides, proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis, 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: Types and Functions, 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 and Types. The most crucial element of the detection strategy is a highly specific antibody-antigen reaction.

Components

• Analyte: the molecule of interest ( antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination)
• 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 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: ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus (enzyme-linked immunosorbent assay)
• 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 Chemiluminescence Blotting Techniques
  • 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 Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination/ 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: Types and Functions.
• A substrate Substrate A substance upon which the enzyme acts. Basics of Enzymes is added, which reacts with the label and results in:
  • Color change ( ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus)
  • Luminescence ( chemiluminescence Chemiluminescence Blotting Techniques)
  • Fluorescence (FIA)
  • Radiation Radiation Emission or propagation of acoustic waves (sound), electromagnetic energy waves (such as light; radio waves; gamma rays; or x-rays), or a stream of subatomic particles (such as electrons; neutrons; protons; or alpha particles). Osteosarcoma emission (radioimmunoassay)
• Changes/signals are detected to determine the presence/amount of antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination in a sample. Detection methods may include:
  • Spectrometry (color change)
  • Luminometry (light emission)
  • Gamma counter ( radiation Radiation Emission or propagation of acoustic waves (sound), electromagnetic energy waves (such as light; radio waves; gamma rays; or x-rays), or a stream of subatomic particles (such as electrons; neutrons; protons; or alpha particles). Osteosarcoma)

Variants of ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus

There are 4 major types of ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus, which are variations of the general immunoassay process:

• Direct ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus:
  • The plate is coated with an antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination.
  • Incubation Incubation The amount time between exposure to an infectious agent and becoming symptomatic. Rabies Virus with a specific, labeled antibody
• Indirect ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus:
  • The plate is coated with an antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination.
  • Incubation Incubation The amount time between exposure to an infectious agent and becoming symptomatic. Rabies Virus with a primary, unlabeled antibody (specific to the antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination)
  • Incubation Incubation The amount time between exposure to an infectious agent and becoming symptomatic. Rabies Virus again with a secondary, labeled antibody (specific to the primary antibody)
• Sandwich ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus (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: Types and Functions):
  • An antibody-coated plate is used.
  • The analyte antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination is added to the plate and incubated.
  • Incubated again with a labeled antibody
• Competitive ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus:
  • An antibody-coated plate is used (an antigen-coated plate is used if testing for a specific antibody).
  • Sample target antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination (or antibody) is added.
  • Labeled target antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination (or antibody) is added and then washed.
  • Note: Unlike other ELISA ELISA An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. St. Louis Encephalitis Virus 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: Types and Functions to the infectious agent. Some examples include:

• Microorganism detection:
  • Legionella pneumophila Legionella pneumophila A species of gram-negative, aerobic bacteria that is the causative agent of legionnaires’ disease. It has been isolated from numerous environmental sites as well as from human lung tissue, respiratory secretions, and blood. Legionella/Legionellosis
  • 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 HIV Anti-HIV Drugs
  • 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 burgdorferi Borrelia burgdorferi A specific species of bacteria, part of the borrelia burgdorferi group, whose common name is lyme disease spirochete. Borrelia 
  • Treponema pallidum Treponema pallidum The causative agent of venereal and non-venereal syphilis as well as yaws. Treponema

Cancer detection and monitoring

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

• PSA PSA A glycoprotein that is a kallikrein-like serine proteinase and an esterase, produced by epithelial cells of both normal and malignant prostate tissue. It is an important marker for the diagnosis of prostate cancer. Prostate Cancer
• Cancer antigen-125
• Alpha-fetoprotein Alpha-fetoprotein The first alpha-globulins to appear in mammalian sera during fetal development and the dominant serum proteins in early embryonic life. Hepatocellular Carcinoma (HCC) and Liver Metastases ( AFP AFP The first alpha-globulins to appear in mammalian sera during fetal development and the dominant serum proteins in early embryonic life. Hepatocellular Carcinoma (HCC) and Liver Metastases)
• Carcinoembryonic antigen Carcinoembryonic antigen A glycoprotein that is secreted into the luminal surface of the epithelia in the gastrointestinal tract. It is found in the feces and pancreaticobiliary secretions and is used to monitor the response to colon cancer treatment. Serum Tumor Markers ( CEA CEA A glycoprotein that is secreted into the luminal surface of the epithelia in the gastrointestinal tract. It is found in the feces and pancreaticobiliary secretions and is used to monitor the response to colon cancer treatment. Serum Tumor Markers)

Allergy Allergy An abnormal adaptive immune response that may or may not involve antigen-specific IgE Type I Hypersensitivity Reaction testing

Immunoassays can be used to detect IgE antibodies IgE antibodies An immunoglobulin associated with mast cells. Overexpression has been associated with allergic hypersensitivity. Type I Hypersensitivity Reaction 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 Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination
• Negative result:
  • Suggests no allergy Allergy An abnormal adaptive immune response that may or may not involve antigen-specific IgE Type I Hypersensitivity Reaction to the antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination
  • Does not completely exclude allergy Allergy An abnormal adaptive immune response that may or may not involve antigen-specific IgE Type I Hypersensitivity Reaction to the antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination (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 Digoxin A cardiotonic glycoside obtained mainly from digitalis lanata; it consists of three sugars and the aglycone digoxigenin. Digoxin has positive inotropic and negative chronotropic activity. It is used to control ventricular rate in atrial fibrillation and in the management of congestive heart failure with atrial fibrillation. Its use in congestive heart failure and sinus rhythm is less certain. The margin between toxic and therapeutic doses is small. Cardiac Glycosides
• Theophylline Theophylline A methyl xanthine derivative from tea with diuretic, smooth muscle relaxant, bronchial dilation, cardiac and central nervous system stimulant activities. Theophylline inhibits the 3. Asthma Drugs
• 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 Cyclosporine A cyclic undecapeptide from an extract of soil fungi. It is a powerful immunosupressant with a specific action on T-lymphocytes. It is used for the prophylaxis of graft rejection in organ and tissue transplantation. Immunosuppressants)
• Antibiotics (e.g., amikacin, vancomycin Vancomycin Antibacterial obtained from streptomyces orientalis. It is a glycopeptide related to ristocetin that inhibits bacterial cell wall assembly and is toxic to kidneys and the inner ear. Glycopeptides, gentamicin Gentamicin Aminoglycosides)

Other diagnostic uses

Additional laboratory tests where immunoassays may be utilized include:

• High-sensitivity troponin I Troponin I A troponin complex subunit that inhibits actomyosin ATPase activity thereby disrupting actin and myosin interaction. There are three troponin I subtypes: troponin i1, i2 and i3. Troponin i3 is cardiac-specific whereas troponin i1 and i2 are skeletal subtypes. Troponin i3 is a biomarker for damaged or injured cardiac myocytes and mutations in troponin i3 gene are associated with familial hypertrophic cardiomyopathy. Myocardial Infarction
• Thyroid Thyroid The thyroid gland is one of the largest endocrine glands in the human body. The thyroid gland is a highly vascular, brownish-red gland located in the visceral compartment of the anterior region of the neck. Thyroid Gland: Anatomy function:
  • Thyroid-stimulating hormone Thyroid-stimulating hormone A glycoprotein hormone secreted by the adenohypophysis. Thyrotropin stimulates thyroid gland by increasing the iodide transport, synthesis and release of thyroid hormones (thyroxine and triiodothyronine). Thyroid Hormones (TSH)
  • Free thyroxine Thyroxine The major hormone derived from the thyroid gland. Thyroxine is synthesized via the iodination of tyrosines (monoiodotyrosine) and the coupling of iodotyrosines (diiodotyrosine) in the thyroglobulin. Thyroxine is released from thyroglobulin by proteolysis and secreted into the blood. Thyroid Hormones ( T4 T4 The major hormone derived from the thyroid gland. Thyroxine is synthesized via the iodination of tyrosines (monoiodotyrosine) and the coupling of iodotyrosines (diiodotyrosine) in the thyroglobulin. Thyroxine is released from thyroglobulin by proteolysis and secreted into the blood. Thyroxine is peripherally deiodinated to form triiodothyronine which exerts a broad spectrum of stimulatory effects on cell metabolism. Thyroid Hormones) and triiodothyroonine ( T3 T3 A T3 thyroid hormone normally synthesized and secreted by the thyroid gland in much smaller quantities than thyroxine (T4). Most T3 is derived from peripheral monodeiodination of T4 at the 5′ position of the outer ring of the iodothyronine nucleus. The hormone finally delivered and used by the tissues is mainly t3. Thyroid Hormones)
• 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 A cobalt-containing coordination compound produced by intestinal microorganisms and found also in soil and water. Higher plants do not concentrate vitamin B 12 from the soil and so are a poor source of the substance as compared with animal tissues. Intrinsic factor is important for the assimilation of vitamin B 12. Folate and Vitamin B12
  • Ferritin Ferritin Iron-containing proteins that are widely distributed in animals, plants, and microorganisms. Their major function is to store iron in a nontoxic bioavailable form. Each ferritin molecule consists of ferric iron in a hollow protein shell (apoferritins) made of 24 subunits of various sequences depending on the species and tissue types. Hereditary Hemochromatosis
  • Vitamin D Vitamin D A vitamin that includes both cholecalciferols and ergocalciferols, which have the common effect of preventing or curing rickets in animals. It can also be viewed as a hormone since it can be formed in skin by action of ultraviolet rays upon the precursors, 7-dehydrocholesterol and ergosterol, and acts on vitamin D receptors to regulate calcium in opposition to parathyroid hormone. Fat-soluble Vitamins and their Deficiencies

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 Error Refers to any act of commission (doing something wrong) or omission (failing to do something right) that exposes patients to potentially hazardous situations. Disclosure of Information

• 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: Types and Functions bind BIND Hyperbilirubinemia of the Newborn to molecules that are similar to the analyte → false positive False positive An FP test result indicates that a person has the disease when they do not. Epidemiological Values of Diagnostic Tests/falsely elevated results)
• Autoantibodies Autoantibodies Antibodies that react with self-antigens (autoantigens) of the organism that produced them. Blotting Techniques (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: Types and Functions, 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: Types and Functions) bind BIND Hyperbilirubinemia of the Newborn to the analyte → false negative False negative An FN test result indicates a person does not have the disease when, in fact, they do. Epidemiological Values of Diagnostic Tests/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: Types and Functions (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: Types and Functions bind BIND Hyperbilirubinemia of the Newborn 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: Types and Functions → false positive False positive An FP test result indicates that a person has the disease when they do not. Epidemiological Values of Diagnostic Tests/falsely elevated results)