Immunoassays

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PRINCIPLES, types and APPLICATIONS of
IMMUNOASSAYS

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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• Immunoassay is an assay based on the reaction of
  an antigen with an antibody specific for the
  antigen.
• Immunochemical reactions form the basis for
  sensitive and specific clinical assays known as
  immunoassays.
• In a typical immunoassay, an antibody is used as
  a reagent to detect the analyte (antigen) of
  interest.
• Monoclonal antibodies are used widely as reagents
  in immunoassay techniques.

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• Types of Immunoassays
• Competitive immunoassays
• Noncompetitive immunoassays
• Radio-Immunoassay (RIA)
• Enzyme-Immunoassay (EIA)
• Fluorescence-Immunoassay (FIA)
• Chemiluminescence-Immunoassay (CIA)

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• Radio Immuno assay (RIA)
• Principle
• In practice, competition between radiolabeled and
  unlabeled antigen or antibody in an
  antigen-antibody reaction analytically is used to
  determine the concentration of the unlabeled
  antigen or antibody.
• Radioactive isotopes iodine, 125I and 131I, and
  tritium (3H) are used as labels.

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• Applications
• diagnosis of hormonal disorders, cancers
• therapeutic monitoring of drugs
• useful in biomedical research
• extraction of a wide variety of compounds which
  include peptides, steroid hormones, vitamins,
  drugs, antibiotics, nucleic acids, structural
  proteins, and hormone receptor proteins.

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• diagnosing insulinomas and sex hormone-sensitive
  tumours.
• digitalisation in the management of congestive
  heart failure.
• estimation of vitamins like B2 and folate,
  hormones like insulin, thyroxine,
  tri-iodothyronine, cortisol, testosterone,
  dihydrotestosterone and estrogens, tropic
  hormones like ACTH, FSH, LH, drugs like digoxin
  and digitoxin and antigens like the Australia
  antigen.

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• Enzyme Linked Immunosorbent Assay (ELISA)
• Enzyme Immunoassy (EIA) uses the catalytic
  properties of enzymes to detect and quantify
  immunological reactions.
• Alkaline phosphatase (ALP), horseradish
  peroxidase (HRP), glucose-6-dehydrogenase (G6D),
  and ß-galactosidase are the enzymes most commonly
  used as labels in EIA.
• ELISA is a heterogenous EIA technique.

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• ELISA is a technique that combines the
  specificity of antibodies with the sensitivity of
  enzyme assay.
• ELISA is used to quantify antigen concentration.
• There are two types of ELISA
• Sandwich ELISA
• Competitive ELISA

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• Sandwich ELISA
• It is a type of ELISA in which the test antigen
  is sandwiched between a capture antibody (primary
  antibody) and an enzyme labelled detection
  antibody (secondary antibody).
• The antigen is quantified from the amount of
  coloured product produced by the enzyme.

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• Competitive ELISA
• It is a type of ELISA in which primary antibody
  is coated to a solid phase and equilibrated with
  the standard and the test antigen.
• Then the enzyme labelled immunogen is added which
  will bind to the primary antibody at sites
  unoccupied by the enzyme labelled immunogen.
• The antibody is quantified from the amount of
  coloured product produced by the enzyme.

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• Applications
• determination of small quantities of proteins
  (hormones, antigens, antibodies) and other
  biological substances.
• commonly used pregnancy test for detection of
  human chorionic gonadotrophin (hCG) in urine is
  based on ELISA.
• diagnosis of AIDS.
• detection and estimation of the antibodies
  against HIV, Hepatitis and Toxoplasma markers.

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• Flourescence Immunoassay (FIA)
• Fluoroimmunoassay (FIA) uses a fluorescent
  molecule as an indicator label to detect and
  quantify immunological reactions.
• Antibody tagged with fluorescein isothiocyanate
  is incubated with cells.
• Antibody fixes with cell surface antigens.

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• In time-resolved fluorescence immunoassay, a
  europium chelate label is excited by a pulse of
  excitation light (0.5 µs).
• Long-lived fluorescence emission from the label
  is measured after a delay (400-800 µs).

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• Applications
• Widely used to measure drugs and other analytes
  (drug metabolites and protein-based substances
  such as bilirubin).
• Most common infectious organisms are identified
  Candida albicans, Haemophilus influenzae,
  Neisseria gonorrhoeae, Shigella, Staphylococcus
  aureus, and several viruses, including rabies
  virus and many enteroviruses.

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• Chemiluminescence immunassay (CLIA)
• Principle
• Chemiluminescence is the light emission produced
  during a chemical reaction.
• In a CLIA, a chemiluminescent molecule is used as
  an indicator label to detect and quantify
  immunological reactions.
• Isoluminol and acridinium esters are the most
  important example of chemiluminescent labels used
  in chemiluminescent immunoassay.

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• Oxidation of isoluminol by hydrogen peroxide in
  the presence of a catalyst (e.g.,
  microperoxidase) produces a relatively long-lived
  light emission at 425 nm.
• Oxidation of an acridinium ester by alkaline
  hydrogen peroxide in the presence of a detergent
  (e.g., Triton X-100) produces a rapid flash of
  light at 429 nm.
• Acridinium esters are high specific activity
  labels (detection limit for the label is 800
  zeptomoles) that can be used to label both
  antibodies and haptens.

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Thank You