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Modified elisa
 

Modified elisa

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    Modified elisa Modified elisa Presentation Transcript

    • ELISAFAYYAZ MUHAMMAD ALI
    • ENZYME LINKEDIMMUNOSORBENTASSAY
    • INTRODUCTION TO ELISA ELISA, or enzyme-linked immunosorbent assay,are quantitative immunological procedures inwhich the Ag- Ab reaction is monitored byenzyme measurements.
    • Why known as ......?Enzyme Linked Immunosorbent Assay1. 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, usuallycoloured.
    • 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 thepresence of Ag : Ab binding. An ELISA can be used to detect either the presenceof Antigens or antibodies in a sample depending howthe test is designed.
    • SubstratePrimaryantibodySecondaryantibodyDifferent antigens in sampleColouredproduct
    • 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 serumwhich gives a positive reaction in the test
    • Basic Steps OfEnzyme-LinkedImmunosorbantAssay
    • ANTIGEN (Ag) Any molecule that induces production of antibodieswhen introduced in the body of an animal is calledantigen.OR any “thing”, foreign to the immune system. e.g.bacteria, viruses, (or their parts), pollen, etc. Protein molecule Carbohydrate molecule. Microorganisms Allergens. Viruses Etc.SYMBOL FOR ANTIGEN
    •  ANTIBODY ( Ab) Antibody: proteins produced by the immunesystem which help defend against antigensSYMBOL FORANTIBODYY
    • Materials Needed Testing sample Antibody (1st, 2nd) / Antigen Polystyrene microtiter plate Blocking buffer Washing buffer Substrate Enzyme
    • Specimen Sample For ELISASERUMCSFSPUTUMURINESEMENSUPERNATANT OF CULUTRESTOOL
    • PRINCIPLE OF INSTRUMENT
    • TYPES OF ELISA Solid phase immunoassay Enzyme Label Competitive assay Non-competitive assay
    • Comparison between Indirect Sandwich & Competitive ELISAto detect Ab (HIV, HCV)to detect Ag ( Tumor Markers, Hormones )to detect Ag ( Free Testosterone)
    • Results
    • Enzymes Used in Elisa Horseradish peroxidase (most commonly used) Alkaline Phosphatase β-galactosidase Lactoperoxidase Tetra Methyl benzidine
    • What Are TheReagents?AndWhat FunctionDo TheyPerform?Antigen: Elisa plate coated with the A60antigen-antibodies complexes.Primary antibody: Human Serum IgG, IgA, &IgMSecondary antibody: Peroxidase-labelled anti-human IgA, IgG, or IgM antibodies that bindto the antibobdy complexes .Enzyme substrate: 3,3’,5,5’ –tetramethylbenzidine (TMB) – a colorlesssolution that when oxidized by HRP turnsblue.Stop Solution: Sulphuric Acid 0.5N (H2So4)
    • Elisa PlateMicrotitre wellsGenerally 96wellsMarked on oneside alphabeticallyNumerically onthe other sideComes with thekit
    • Measures the absorbanceat 450nm With the help ofELISA READER.Calculate the absorbancefor each sample andreference.ELISA PLATE READYFOR READING
    • ELISA READERTHERMOLAB SYSTEM(USA)
    • WHAT IS CUT OF VALUE?ELISA is semiquantitative method. The calculation is done asfollows. The units of ELISA is OD ratio:Sample value= sample OD/cut-off ODWhat is Cut-off Value ………?
    • APPLICATIONS OF ELISA1- Hormones 7- Vaccine Quality Control2- Proteins 8- FOR GMO (Genetically modifiedorganism)3- Infectious Agent ( Viral, Bacterial,Parasitic, Fungal )9- For Rapid Test4- Drug Markers 10- IgG, IgM, IgA5- Tumor Markers 11- In New Born Screening6- Serum Proteins 12- In Clinical Research
    • THANKYOU