Immunoassay

presentation

1. PRESENTATION ON IMMUNOASSAY Presented by: : Rabiya Ahsan M.Pharm ( pharmacology ) Integral university 2018-2019 1

2. General principle of immunoassay. 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

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4. Competitive immunoassay In a 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 are two 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. H omogeneous immunoassays Homogenous methods 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 As in 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 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 OF ELISA U se 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 Q ualitative determines antigen or antibody is present or absent Q uantitative 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)  Any molecule 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 The radioimmunoassay is perhaps the oldest type of immunoassay . Here, a radioisotope is attached to an antigen of interest and bound with its complementary antibody . Then a sample with the antigen to be measured is added . It competes with the radioactive antigen, kicks it out of the binding spot and replaces it . After washing away unbound antigens the radioactivity of the sample is measured. The amount of radioactive signal is inversely related to the amount of target antigen . Which emits radiation that can be measured with a beta or gamma counter 19

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21. . Fluoroimmunoassay In a 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 22

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24. Reference Wild david (ED).2005 . The immunoassay Handbook. Kidlington,oxford:Elsevier https://en.m.Wikipedia.org/wiki/Immunoassay 24

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