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Immunoassay
This presentation provides an overview of immunoassays. It discusses the general principles, including that immunoassays use antibodies and antigens to generate a measurable result. The main types are then summarized: competitive immunoassays where antigens compete for binding sites; non-competitive where binding is directly proportional to the amount of antigen; and homogeneous vs heterogeneous based on whether separation is required. Specific techniques like ELISA, radioimmunoassay, and fluoroimmunoassay are also briefly explained. Applications include disease diagnosis and factors like accuracy, precision, calibration and controls are noted.
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Presentation by Rabiya Ahsan on the topic of immunoassays in pharmacology.
Immunoassay uses antibodies and antigen complexes to generate measurable results.
Includes competitive, noncompetitive, homogeneous, and heterogeneous immunoassays.
In competitive assays, the analyte competes with a labeled antigen, measured in two formats.
Measures labeled analyte proportionally to antigen; includes one-site and two-site formats.
Applied to measure small analytes; no separation is required for bound and free analytes.
In these assays, unlabelled analyte competes with labelled analyte; unbound is removed.
Techniques include ELISA, radioimmunoassay, fluoroimmunoassay, and enzyme immunoassays.
EMI measures drug concentration based on enzyme activity affected by antibody binding.
ELISA uses an enzyme linked to color change for detection of antigen-antibody binding.
Qualitative ELISA determines presence/absence, while quantitative assesses antibody quantity.
Antigens induce antibody production; they include various foreign molecules like bacteria.
Antibodies protect against antigens; main types include IgG, IgD, IgA, IgM, and IgE.
Oldest immunoassay using radioisotopes; measures antigen by competition with radioactive form.
Utilizes fluorescently labeled antibodies; measures intensity of fluorescence for antigens.
Accuracy, precision, calibration, and control are critical for immunoassay efficacy.
Used in diagnosing diseases such as immunodeficiency, autoimmune disorders, and tumors.
References including 'The Immunoassay Handbook' and relevant online resources.
Thankful conclusion from Rabiya Ahsan, appreciation for support and encouragement.
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Immunoassay
- 1. PRESENTATION ON IMMUNOASSAY Presented by:: Rabiya Ahsan M.Pharm (pharmacology) Integral university 2018-2019 1
- 2. General principle ofimmunoassay. • Immunoassay is a test that uses antibodies and antigen complexes as a means of generating a measurable result. • An antibody: antigen complex is also known as an immune complex. • An immunoassay is a test that utilizes immune complexing when antibodies and antigens are brought together 2
- 3. Types. Competitive immunoassay Noncompetitive immunoassay Homogenousimmunoassay Heterogeneous immunoassays 3
- 4. Competitive immunoassay • Ina competitive format , unable analyte (usually the antigen) in the test sample is measured by its ability to compete with the labelled antigen in the immunoassay • Its is less label measured in the means more of the unlabelled( test sample)antigen is present. 4
- 5. • There aretwo version of the competitive format. • One step format • Two step format 5
- 6. Non competitive immunoassay •In noncompetitve assay , the measurement of the labelled anlayte usually the antibody) is directly proportional to the amount of antigen present in the sample • Noncompetitive assay formats can use either one step or two step methods. • In the two step assays format , there are wash steps in which the sandwich binding complex is isolate and wash to remove excess unbound labelled reagent 6
- 7. One-site, noncompetitive immunoassays •The unknown analyte in the sample binds with labelled antibodies. • • The unbound, labelled antibodies are washed away, and the bound, labelled antibodies are measured • The intensity of the signal is directly proportional to the amount of unknown analyte . 7
- 8. Two-site, noncompetitive immunoassays •The analyte in the unknown sample is bound to the antibody site, then the labelled antibody is bound to the analyte. • The amount of labelled antibody on the site is then measured. • It will be directly proportional to the concentration of the analyte because the labelled antibody will not bind if the analyte is not present in the unknown sample. • This type of immunoassay is also known as a sandwich assay as the analyte is "sandwiched" between two antibodies. . 8
- 9. Homogeneous immunoassays • Homogenousmethods have been generally applied to the measurement of small analytes such as abused and therapeutics drugs. • since homogenous methods do not require the separation of the bound Ab- Ag* from the free Ag*, there are generally much easier and faster to perform 9
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- 11. Hetrogenous immunoassay • Asin a competitive, heterogeneous immunoassay, unlabelled analyte in a sample competes with labelled analyte to bind an antibody • the labelled, unbound analyte is separated or • washed away, and the remaining labelled, bound analyte is measured. 11
- 12. TECHNIQUESOF IMMUNOASSAY ELISA Radioimmunoassay Fluoroimmunoassay Enzyme immunoassaywith enzyme labelled EMI 12
- 13. Enzyme Multiplied immunoassay(EMI) • EMI: the drug in the sample and the labelled with G6PD compete for antibody binding sites • Binding inhibit enzyme activity , while free enzyme remains activity to interact with the substrate • Enzyme activity absorbance is directly proportional to drug concentration 13
- 14. BASIC PRINCIPLE OFELISA • Use an enzyme to detect the binding of antigen (Ag)antibody (Ab). • The enzyme converts a colorless substrate(chromogen) to a colored product, indicating the presence of Ag : Ab binding. • An ELISA can be used to detect either the presence of Antigens or antibodies in a sample depending how the test is designed. • ELISA was developed in 1970 and became rapidly accepted ELISA 14
- 15. ELISA Qualitative/Quantitative Qualitative determines antigenor antibody is present or absent Quantitative determines the quantity of the antibody The highest dilution of the specimen usually serum which gives a positive reaction in the test 15
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- 17. ANTIGEN (Ag) • Anymolecule that induces production of antibodies when introduced in the body of an animal is called antigen. • OR any thing, foreign to the immune system. e.g. • bacteria, pollen, • Protein molecule • Carbohydrate molecule. • Microorganisms • Allergens. • Viruses Etc. 17
- 18. ANTIBODY ( Ab) Antibody:proteins produced by the immune system which help defend against antigens. Types of antibody are ; • IgG, • IgD, • IgA, • IgM • ,IgE 18
- 19. RADIOIMMUNOASSAY 1. The radioimmunoassayis perhaps the oldest type of immunoassay. 2. Here, a radioisotope is attached to an antigen of interest and bound with its complementary antibody. 3. Then a sample with the antigen to be measured is added. 4. It competes with the radioactive antigen, kicks it out of the binding spot and replaces it. 5. After washing away unbound antigens the radioactivity of the sample is measured. 6. The amount of radioactive signal is inversely related to the amount of target antigen. 7. Which emits radiation that can be measured with a beta or gamma counter 19
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- 21. . Fluoroimmunoassay • Ina fluoroimmunoassay the antibodies are labeled with fluorescent probes. • After incubation with antigen the antibody-antigen complexes are isolated and the fluorescent intensity is measured. 21
- 22. Factors impacting immunoassays Accuracy andprecision Calibration and control 22
- 23. Application of immunoassay Diagnosisof diseases Immunodeficiency disease Autoimmune disease Hypersensitivity tumour 23
- 24. Reference • Wild david(ED).2005 .The immunoassay Handbook. Kidlington,oxford:Elsevier • https://en.m.Wikipedia.org/wiki/Immunoassay 24
- 25. THANKYOU Thanks for alwayslistening to me , supporting me , and encouraging me &THANKYOU SO MUCH 25