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Edit Comment Close Premium member Presentation Transcript Slide 2: Application of ELISA Test SeminarSlide 3: By: Shahid Nawaz 2010-ag-1064 Supervisor: Prof. Dr.Tanwir Ahmad MalikELISA: ELISA ELISA …… Enzyme-linked immunosorbent assay It is a biochemical technique used to detect the presence of an antibody or an antigen in a sample . An enzyme linked immunosorbent assay (ELISA) is a test performed to determine the levels of protein in a biological sampleWhy known as ......? Enzyme Linked Immunosorbent Assay: Why known as ......? Enzyme Linked Immunosorbent Assay Antigen of interest is absorbed on to plastic surface (‘ sorbent ’). 2. Antigen is recognised by specific antibody (‘ immuno ’). 3. This antibody is recognised by second antibody (‘ immuno ’) which has enzyme attached (‘ enzyme-linked ’). 4. Substrate reacts with enzyme to produce product, usually coloured.HISTORY: HISTORY Prior to the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960 The term ELISA was first used by Engvall & Perlma in 1971.How does an ELISA test work?: How does an ELISA test work? There are variations of the ELISA test, but the most basic type consists of an antibody attached to a solid surface. This antibody has affinity for (will latch on to) the substance of interest For example……… human chorionic gonadotropin (HCG), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bindThe more HCG which is present in the test sample, the less . enzyme linked HCG will bind.BASIC PRINCIPLE OF ELISA: BASIC PRINCIPLE OF ELISA 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.Slide 9: Substrate Primary antibody Enzyme Secondary antibody Different antigens in sample Coloured productELISA Qualitative/Quantitative: ELISA Qualitative/Quantitative Qualitative determines antigen or antibody is present or absent Quantitative determines the quantity of the antibody Titer The highest dilution of the specimen usually serum which gives a positive reaction in the testVARIANTS OF ELISA: VARIANTS OF ELISA DAS-ELISA : Double Antibody Sandwich also called Direct ELISA DAC-ELISA: Direct Antigen-coated ELISA also called Indirect ELISA TAS-ELISA: Triple Antibody Sandwich ELISASlide 12: DAS ELISAMATERIALS: MATERIALS ELISA plates Antiserum Buffers Micropipettes (10-100ul and 100-1000ul) 200ul micropipette (fixed) or adjustable multichannel micropipette Micropipette tips Distilled or purified water Plate Washer Plate Dryer Electric Balance Washing squeeze bottle Magnetic bars Electric stirrer Humidity chamber (plastic box with moist tissue papers) Mortar and Pestle ELISA Reader & PrinterGENERAL APPARATUS: GENERAL APPARATUSSlide 15: MethodologyBUFFERS USED IN ELISA: BUFFERS USED IN ELISA Coating Buffer : Dissolve in distilled water to 1000 ml: Sodium carbonate (anhydrous) 1.59 g Sodium bicarbonate 2.93 g Sodium azide 0.2 g pH 9.6 Washing Buffer : Dissolve in distilled water to 1000 ml: Sodium chloride 8.0 g Sodium phosphate, dibasic 1.15g Potassium phosphate, monobasic 0.2 g Potassium chloride 0.2 g Tween-20 0.5 g pH 7.4BUFFERS USED IN ELISA: BUFFERS USED IN ELISA Extraction Buffer : Dissolve in 1000 ml of 1X PBST: Sodium sulfite (anhydrous) 1.3 g Polyvinylpyrrolidone (PVP) 20.0 g Sodium azide 0.2 g Powdered egg (chicken) albumin 2.0 g Tween-20 20.0 g pH 7.4 Enzyme Conjugate Buffer : Add to 1000 ml 1X PBST: Agar albumin 2.0 g Polyvinylpyrrolidone (PVP) 20.0 g Sodium azide 0.2 g pH 7.4BUFFERS USED IN ELISA: BUFFERS USED IN ELISA Substrate Buffer : Dissolve in 800 ml distilled water: Magnesium chloride hexahydrate 0.1 g Sodium azide 0.2 g Diethanolamine 97.0 ml pH 9.8 Reaction Stopping Solution : NaOH 3 MDIFFERENT STEPS OF DAS-ELISA: DIFFERENT STEPS OF DAS-ELISASlide 20: DAS-ELISAANTIBODY COATING STEP:: Loading Coating Antibody ANTIBODY COATING STEP:INCUBATION: INCUBATION Incubate the coating antibody loaded plate at 4 o C for 24 hoursWASHING:: Plate Washer WASHING:DRYING:: DRYING: Plate DryersSAMPLING:: SAMPLING: Test Tube Plants Glass House Potted PlantsEXTRACTION:: EXTRACTION: Weighing Balance Grinding & Diluting SampleSAMPLE LOADING:: SAMPLE LOADING: Plant Extract Loaded PlateSlide 28: Incubation (at 4 o C for 24 hours) Washing & Drying Loading Enzyme Conjugated Antibody Incubation (at 4 o C for 24 hours) Washing & Drying Loading PNP Substrate Incubation (at 37 o C) Stopping ReactionVIRUS DETECTION:: VIRUS DETECTION: ELISA Reader PrinterAdvantages of ELISA: Advantages of ELISA ELISA tests are generally highly sensitive and specific and compare to radioimmune assay (RIA) tests. They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus). Reagents are relatively cheap & have a long shelf life ELISA is highly specific and sensitive No radiation hazards occur during labelling or disposal of waste. Easy to perform and quick procedures Equipment can be inexpensive and widely available. ELISA can be used to a variety of infections.Disadvantage of ELISA: Disadvantage of ELISA It requires skilled laboratory technicians and specialized laboratory equipment. Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes. Enzyme activity may be affected by plasma constituents. Kits are commercially available, but not cheap Very specific to a particular antigen. Won’t recognize any other antigen False positives/negatives possible, especially with mutated/altered antigenAPPLICATIONS: APPLICATIONSApplications….: Applications…. Enzyme Linked Immunosorbant Assays (ELISA) have been used for decades in research and development, disease diagnosis and other critical fields in sciences.Cont……: Cont…… Detecting infections sexually-transmitted agents like HIV, syphilis and chlamydia hepatitis B and C Toxoplasma gondii Detecting allergens in food and house dust Measuring toxins in contaminated foodCont….: Cont…. ELISA is used to detect many bacterial and viral antigens, including human immunodeficiency virus (HIV), malaria, cholera, measles, and mumps The ELISA has been used as a diagnostic tool in medicine and plant pathology , as well as a quality control check in various industriesSlide 36: Diagnosis and identification of viruses and prokaryotes Strain differentiation Serological relationship , close or distant, and virus classification Seed-borne viruses Resistance breeding Production of virus free material Monitoring of viruliferous vectors, individual insects and IPM strategy Component of quarantine department Also used for the identification of proteins, fungi, bacteria You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.