ELISA by MR Zeshan Ul HAAQ FROM uaf,.pptx

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1. Muhammad Zeeshan-Ul-Haq 2018-ag-1883 M.Phil CMS ELISA

2. It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens in blood. A number of enzymes have been used for ELISA such as alkaline phosphatase, horse radish peroxidase and beta galactosidase . Enzyme-Linked Immuno-Sorbent-Assay.

3. ELISA is process, combination of specificity of antibodies with sensitivity of enzymes. It provides antibody and antigen concentration. Based on variable method, antigens can be detect by antibody and antibodies is used to recognized antigen. Color product produced to measure function and quantity of antigen and antibodies, To detect antibodies, hormones, proteins, peptides and antigens Principle

4. On the base of binding structure between antigen & antibody: 1) Indirect ELISA 2)Sandwich ELISA 3)Direct ELISA 4) Competitive ELISA The indirect (to detect antibodies in the sample) and the sandwich (to detect antigens in the sample) ELISA methods are the two most common types used. Types of ELISA method

5. Binding Known Antigen : known antigen being bound to the wells of a microtiter plate. Blocking - The other unoccupied sites are then bound by a concentrated solution of non-interacting protein, like casein or bovine serum albumin, to prevent other proteins from adhering. Washing – to remove any unbound antigen and non-interacting protein. Adding Primary Antibody - serum containing the primary antibodies is added. Antibodies could be HIV, rabies, or hepatitis B antibodies. Washing – Rinse to remove any antibodies that did not bind to the known antigen . Indirect ELISA

6. f ) Adding Enzyme-linked Secondary Antibody - added next to bind to antibodies. The enzyme on the secondary antibodies are proteins, such as horse radish peroxidase or alkaline phosphatase. g) Washing – Rinse to remove any secondary antibodies that did not bind to the primary antibody. h) Adding Substrate( chromogen ) - A substrate is then applied which is converted by the enzyme to give color product. e.g. horse radish peroxidase. Alkaline phosphatase, turns yellow. i ) Reading Results - Spectrophotometer,electrochemical device. C ontinu …

7. Antibody is coated on microtiter well Antigen containing sample as added and form antigen-antibody complex Wash it and add second antibody and allowed to react to form complex Unbound secondary antibody is removed by washing Then add substrate to hydrolyzed by enzyme to produce a color product. Sandwich ELISA

8. Antibody is incubated with sample containing antigen. Antigen-antibody complex are added to the microtitre well which are pre-coated with the antigen. Wash the plate to remove unbound antibody. Enzyme linked secondary antibody which is specific to the primary antibody is added. Wash the plate, so that unbound enzyme-linked antibodies are removed. Add substrate which is converted by the enzyme into a fluorescent signal. Competitive ELISA

9. Antigen is absorbed to plate Block all binding sites by protein as serum of bovine albumin Add enzyme linked antibody, forming a complex Washed unbound antigens Added substrate and it form color product Direct ELISA

10. Presence of antibody and antigen in sample Determination of serum antibody concentration in virus test Diagnostic procedures for detecting infection. To detect food allergens in food industry To track spread of disease it applied in disease outbreaks e.g. HIV, bird flu, common cold etc. Applications

11. Thank You