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Immunoassay for Pharmacology

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Immunoassay for MD Pharmacology Students

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Immunoassay for Pharmacology

  2. 2. INTRODUCTION  An immunoassay is a specific type of biochemical test that measures the presence or concentration of a substance (referred to as the "analyte") in solutions that frequently contain a complex mixture of substances.
  3. 3. INTRODUCTION  Antibody/Antigen reaction provides the means of generating a measurable result.  “Immuno” refers to an immune response that causes the body to generate antibodies.  “Assay” refers to a test.  An immunoassay is a test that uses immunocomplexing when antibodies and antigens are brought together.
  4. 4. INTRODUCTION  An antibody is a protein produced in the body to a foreign substance.  An antigen is the substance that the body is trying to eliminate by mounting an immune response.  An analyte is anything measured by a laboratory test.  Immunoassays may measure either the antigen or antibody.  Immunoassays use one or more select antibodies to detect analytes of interest. LABEL 
  5. 5. LABEL   All immunoassays require the use of labeled material in order to measure the amount of antigen or antibody present. A label is a molecule that will react as part of the assay, so a change in signal can be measured in the blood:reagent solution. EXAMPLES
  6. 6. Examples of a label    a radioactive compound, an enzyme that causes a change of color in a solution, or a substance that produces light.
  7. 7. Categories of Immunoassay  Competitive  Noncompetitive  Labels may be applied to either the antibody.. Homogeneous ..or the antigen.  Heterogeneous
  8. 8. Competitive Assays   In a competitive format, unlabeled analyte (usually the antigen) in the test sample is measured by its ability to compete with the labeled antigen in the immunoassay. In a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present. • There are two versions of the competitive format: • One Step format • Two step format
  9. 9. One step competitive format  In the one step competitive format , both the labeled antigen reagent (Ag*) and the unlabeled specimen (or test sample analyte) compete for a limited amount of antibody.
  10. 10. Two step competitive format    In the two step competitive format, the antibody concentration of the reaction solution is present in excess in comparison to the concentration of antigen. Antibody reagent is first incubated with specimen containing antigens of interest; then in the second step, labeled antigen is added. More sensitive than one step.
  11. 11. Noncompetitive Assays  Noncompetitive assay formats give the highest level of sensitivity and specificity.  They are normally used to measure critical analytes such as cardiac and hepatitis markers. • In noncompetitive assays, the measurement of the labeled analyte (usually the antibody) is directly proportional to the amount of antigen present in the sample.
  12. 12. Enzyme Immunoassay (EIA)  In enzyme immunoassays (EIA), enzyme labels are used.  Typical enzyme labels include alkaline phosphatase, horseradish peroxidase and galatosidase.  EIA tests typically use a change in color, emmission of light or other signal.
  13. 13.  A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
  14. 14. Radioimmunoassay (RIA)  Radioimmunoassay (RIA) techniques were developed in the 1960s and use radioactive isotopes as a label
  15. 15. Radioimmunoassay (RIA)       Very sensitive in vitro assay technique Less expensive Radioactive substances are used Commonly used radioisotope is I 125 Micro curies of radioactivity – minimum radiation Micro-gram and Pico-gram quantities of substances can be analyzed.
  16. 16. RAST      Radioallergosorbent test Modification of RIA Antigen attached to plate well – allergen Radio labeled ligand used will attach only to IgE antibody – specific for allergen. Used to detect specific allergen in persons suspected to be suffering from Type I hypersensitivity.
  17. 17. Fluorescence Polarization Immunoassay (FPIA)  Homogeneous competitive fluoresence immunoassay.  With competitive binding, antigen from the specimen and antigenfluorescein (AgF) labeled reagent compete for binding sites on the antibody. FPIA is used to provide accurate and sensitive measurements of small toxicological analytes such as therapeutic drugs and drugs of abuse. 
  18. 18. Fluorescence Polarization Immunoassay  FPIA uses three concepts to measure specific analytes in a homogeneous format:  Fluorescence  Rotation of molecules in solution  Polarized light
  19. 19. Fluorescence  Fluorescein is a fluorescence label that absorbs light at 490 nm and releases this energy at 520 nm.  Larger molecules rotate more slowly in solution that smaller molecules.  Because of this, we can distinguish between the smaller antigenfluorescein (AgF) label from antibody bound antigen-fluorescein (AbAgF).
  20. 20. Surround Optical Fiber Immunoassay (SOFIA)     an ultra-sensitive, in vitro diagnostic platform incorporating a surround optical fiber assembly that captures fluorescence emissions from an entire sample. extremely high limit of detection , sensitivity and dynamic range. sensitivity is measured at the attogram level (10−18g), making it approximately one billion times more sensitive than conventional diagnostic techniques.
  21. 21. Surround Optical Fiber Immunoassay (SOFIA)   ability to detect naturally occurring prions in the blood and urine of disease carriers first reliable ante mortem screening test for vCJD, scrapie and other transmissible spongiform encephalopathies
  22. 22. Magnetic immunoassay      novel type of diagnostic immunoassay using magnetic beads as labels involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of the pair. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte (virus, toxin, bacteria, cardiac marker,etc.) quantity in the initial sample.
  23. 23. References    Text book of Biochemistry for medical students by DM Vasudevan Textbook of Microbiology, Annanth narayan. Wikipedia
  24. 24. THANK YOU.
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Immunoassay for MD Pharmacology Students


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