IMMUNOASSAYS.pptx

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1. IMMUNOASSAYS Dr. Sherzad M Taher Medical Immunology

2. Definition Immunoassay – two words ; immuno - and assay Immuno - representing immune, immunity, or immunology in compound words Assay is an investigative (analytic) procedure in laboratory for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity (the analyte ). What is immunoassay? Immunoassay is a biochemical method that uses the specificity of an antigenantibody reaction to detect and quantify target molecules in biological samples.

3. The principles of immunoassays The immunoassay will use those highly specific antibodies to probe for molecules of interest when they are in mixtures with other molecules

4. Types of Immunoassays : 1 .Enzyme Immunoassays (EIA) e.g Enzyme-linked immunosorbent assays (ELISA) 2 .Radioimmunoassay (RIA) 3 .Counting Immunoassay (CIA) 4 .Fluoroimmnoassay (FIA) 5 .Chemiluminescenceimmunoassay(CLIA)

5. 1 . Enzyme immunoassay An enzyme immunoassay is any of several immunoassay methods that use an enzyme bound to an antigen or antibody. These may include: Enzyme-linked immunosorbent assay (ELISA)

6. What is ELISA? Enzyme-linked immunosorbent assay is a biomolecular technique that utilizes the specificity of an antibody, as well as the sensitivity of enzyme assays, to detect and quantify molecules such as hormones, peptides, antibodies , and proteins.

7. ELISA Components • Coated Plates- The 96-well plates are made of polystyrene and coated with either inactivated antigen or antibody. This coating is the binding site for the antibodies or antigens in the sample • Sample Diluent- Most assays require a specific dilution of the sample • Controls- Controls are also used to validate the assay and to calculate sample results. • Conjugate- ELISA conjugates are enzyme-labeled antibodies or antigens that react specifically to plate-bound sample analytes . • Substrate – e.g For peroxidase conjugates, the substrate is a mixture of hydrogen peroxide and a chromogen that reacts with the enzyme portion of the conjugate to produce color. • Wash Concentrate -a buffered solution containing detergent used to wash away unbound materials from the plates. • Stop Solution- The stop solution stops the enzyme-substrate reaction and, thereby, the color development.

8. ELISA KIT

9. ELISA READER

10. Methods of ELISA Direct ELISA Refers to an ELISA in which only a labeled primary antibody is used when the presence of an antigen is analyzed

11. Indirect ELISA Refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labeled secondary antibody

12. Sandwich ELISA The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody

13. Competitive ELISA Also known as inhibition ELISA The central event of competitive ELISA is a competitive binding process executed by original antigen (sample antigen) and add-in antigen.

14. Applications of ELISA • detection of Mycobacterium antibodies in tuberculosis • detection of rotavirus in feces • detection of hepatitis B markers in serum • detection of hepatitis C markers in serum • detection of enterotoxin of E. coli in feces • detection of HIV antibodies in blood samples • detection of SARS-CoV-2 antibodies in blood samples

15. 2. RADIOIMMUNOASSAY (RIA) It is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, (for example, hormone levels in blood) by use of antibodies.

16. 3. Counting Immunoassay (CIA) An application of particle counting technology to serological tests . • Latex particles are agglutinated by antibodies or antigens of interest and are quantified by scattered laser light while passing through a controlled sheath flow, which is also used in flowcytometry .

17. Applications of Counting Immunoassay The detection of hepatitis B virus (HBV) antigen antibodies, Anti-adult T-cell leukemia antibodies A ntitoxoplasma antibodies , hormones , and serum acute-phase proteins.

18. 4.Chemiluminescence Immunoassay (CLIA) Chemiluminescence immunoassay (CLIA) is an assay that combine chemiluminescence technique with immunochemical reactions. Similar with other labeled immunoassays (RIA, FIA, ELISA), CLIA utilize chemical probes which could generate light emission through chemical reaction to label the antibody. CLIA utilize chemical probes which could generate light emission through chemical reaction to label the antibody

19. 5 . Fluoroimmunoassay (FIA) uses as the detection reagent;a fluorescent compound which absorbs light or energy (excitation energy) at a specific wavelength and then emits light or energy at a different wavelength . It is sometimes more sensitive than RIA .

20. References https://en.wikipedia.org/wiki/Assay • https://en.wikipedia.org/wiki/Immunoassay • https://www.news-medical.net/life-sciences/What-are-Immunoassays.aspx • https://www.sciencedirect.com/topics/neuroscience/immunoassay • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614608/ • https://www.lornelabs.com/news-events/blog/what-is-chemiluminescentimmunoassay • https://en.wikipedia.org/wiki/Radioimmunoassay • https://www.sciencedirect.com/topics/nursing-and-healthprofessions/fluoroimmunoassay • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC516346/