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ELISA tests are usually performed in microwell plates. The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. INTRODUCTION TO ELISASlide 3: Antibody is immobilized on micro-plate wells Competition between in sample and labeled enzyme for antibody binding sites The unbound material is washed out Chromogenic substrate added to develop color Resulting color is read in a spectrophotometer PRINCIPLE OF ELISASlide 4: Antibodies (also known as immunoglobulins abbreviated Ig ) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. DefinitionsSlide 5: Light chain Heavy chain Disulfide bonds Antigens Antibody StructureSlide 6: Immune Response A. Pathogen C. Macrophage D. Macrophage E. Macrophage F. T cell B. B cells G. B cell H. Memory B cells I. Plasma cells J. Antibodies attach to pathogen HIV Antibodies are ineffective Against HIVSlide 7: Antigens A substance that when introduced into the body stimulates the production of an antibody Immunoassay A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sampleSlide 8: Analyte The sample being analyzed and in immunoasssays the analyte is either Antibody or AntigenSlide 9: Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The technique is divided into 1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA ELISA TECHNIQUESlide 11: ELISA PLATESlide 12: The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal). Competitive ELISASlide 14: The ELISA plate is coated with Antibody to detect specific antigen SANDWICH ELISASlide 15: Prepare a surface to which a known quantity of capture antibody is bound. Block any non specific binding sites on the surface Apply the antigen-containing sample to the plate.Slide 16: Wash the plate, so that unbound antigen is removed. Apply enzyme linked primary antibodies as detection antibodies which also bind specifically to the antigen. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.Slide 17: Apply a chemical which is converted by the enzyme into a coloured product. Measure the absorbency of the plate wells to determine the presence and quantity of antigenSlide 19: The protein antigen to be tested for is added to each well of ELISA plate, where it is given time to adhere to the plastic through charge interactions A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen INDIRECT ELISASlide 20: Then the serum is added, which contains a mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well. Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to itSlide 21: A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. Often a spectrometer is used to give quantitative values for colour strengthSlide 24: Screening donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) hepatitis C (presence of antibodies) hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function) APPLICATIONSlide 25: Detecting infections sexually-transmitted agents like HIV, syphilis and chlamydia hepatitis B and C Toxoplasma gondii Detecting allergens in food and house dust Measuring "rheumatoid factors" and other autoantibody in autoimmune diseases like lupus erythematosus Measuring toxins in contaminated food Detecting illicit drugs, e.g., cocaine opiatesSlide 26: A newer technique uses an solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents. ELISA Reverse method & deviceSlide 27: The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays. The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples. One ogive is left unsensitized to measure the non-specific reactions of the sample. The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits. ADVANTAGESSlide 28: THANK YOU!! You do not have the permission to view this presentation. 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