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IMMUNOASSAYS

BIOCHEMISTRY

mprasadnaidu
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IMMUNOASSAYS

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  1. PRINCIPLES, types and APPLICATIONS of IMMUNOASSAYS M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

  2. 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.

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

  4. 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.

  5. 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.

  6. 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.

  7. 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 β-galactosidase are the enzymes most commonly used as labels in EIA. • ELISA is a heterogenous EIA technique.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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).

  14. 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.

  15. 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.

  16. 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.

  17. Thank You

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