Assays and applications of immune response(neutralization and precipitation)
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Assays and applications of immune response(neutralization and precipitation) Presentation Transcript

  • 1. 11 May 2002
    1
  • 2. ASSAYS AND APPLICATIONS OF IMMUNE RESPONSE
    PRESENTED BYBILAL ANJUM BUTT 50-E
    11 May 2002
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  • 3. IMMUNE ASSAYS
    Immunoassays are a group of sensitive analytical tests that utilize very specific antibody/antigen complexes to produce a signal that can be measured and related to the concentration of a compound in solution.
    TYPES
    Radioimmunoassays(RIAs)
    Fluorescent Polarized Immunoassay
    Enzyme Multiplied Immunoassay (EMIT)
    Enzyme linked immunosorbant assay (ELISA)
    11 May 2002
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  • 4. ELISA(Enzyme-linked immunosorbent assay)
    11 May 2002
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  • 5. INTRODUCTION TO ELISA
    ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique involving the reaction of antigen and antibody in vitro. ELISA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. ELISA tests are usually performed in microwell plates.
    It is a sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein.
    11 May 2002
    5
  • 6. HISTORY
    It is first described by Peter Perlmannand Eva Engval , and Anton SchuursandBauke van Weemen.
    The technique was subsequently developed by Voller and col. using microplates, that permitted the development of highly sensitive and accurate kits.
    11 May 2002
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  • 7. COMPONENTS OF AN ELISA
    Antibody: IgG fraction of serum
    Enzyme:Peroxidase from horseradish,Alkalinephosphatase from E. coli,β-galactosidase from E coli.,Glucoseoxidase.
    Substrate: TMB (3,3',5,5', tetramethylbenzidine).
    11 May 2002
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  • 8. PRINCIPLE OF ELISA
    The sample with an unknown amount of antigen is immobilized on a solid support.
    The detection antibody is added ,forming a complex with antigen.
    The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme.
    Between each step the plate washed with a mild detergent solution.
    After the final wash step, adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
    11 May 2002
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  • 9. 8. Observe colour development
    7. Add substrate for enzyme
    1. Add antigen
    2. Wash with PBST
    6. Wash with PBST
    4. Wash with PBST
    3. Add primary antibody
    5. Add secondary antibody
    11 May 2002
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  • 10. ELISA
    Step 1
    inactivated HIV antigens
    Step 2
    serum antibodies
    Step 3
    Anti-human Ig
    coupled to enzyme
    Step 4
    Chromogen or substrate
    Step 5 ( Measurement )
  • 11. CRITERIA FOR CHOICE OF A MARKER ENZYME
    Should be easily coupled to ligands & the labelled complex must be stable.
    The reactivity should be retained after linking of the enzyme to the antigen/antibody.
    The chosen enzymes should not be normally present in the patient samples.
    11 May 2002
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  • 12. TYPES OF ELISA
    INDIRECT ELISA
    SANDWICH ELISA
    COMPETETIVE ELISA
    11 May 2002
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  • 13. INDIRECT ELISA
    The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since first the antigen is coated and specific antibody is added which forms complex.Then add enzyme-conjugated secondry antibody and add substrate and measure colour.
    11 May 2002
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  • 14. ADVANTAGES AND DISDVANTAGES
    Advantages of indirect detection
    Wide variety of labeled secondary antibodies are available commercially.
    Sensitivity is increased because each primary antibody contains several sites that can be bound by the labeled secondary antibody, allowing for signal amplification.
    Secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect
    Disadvantages of indirect detection
    Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.
    An extra incubation step is required in the procedure.
    11 May 2002
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  • 15. SANDWICH ELISA
    Plate is coated with a antibody.
    Sample is added, and any antigen present binds to antibody.
    Detecting antibody is added, and binds to antigen.
    Enzyme-linked secondary antibody is added, and binds to detecting antibody.
    Substrate is added, and is converted by enzyme to detectable form.
    11 May 2002
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  • 16. Competitive binding assay
    Unlabeled antibody is incubated in the presence of its antigen.
    These bound antibody/antigen complexes are then added to an antigen coated well.
    The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
    The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
    A substrate is added and check the colour change.
    11 May 2002
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  • 17. Advantages
    The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.
    11 May 2002
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  • 18. Immunologic tests: Types of ELISA’s…
    11 May 2002
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  • 19. APPLICATIONS
    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)
    11 May 2002
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  • 20. Detecting infections
    sexually-transmitted agents like HIV, syphilis and chlamydia
    hepatitis B and C
    Toxoplasmagondii
    Detecting allergens in food and house dust
    Measuring toxins in contaminated food
    Detecting illicit drugs, e.g.,
    cocaine
    opiates
    11 May 2002
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  • 21. ELISA Reader
    11 May 2002
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  • 22. ADVANTAGES OF ELISA
    • Sensitive assay
    • 23. Equipments are widely available.
    • 24. No radiation hazards.
    • 25. Reagents are cheap with long shelf life.
    • 26. Adaptable to automation and high speed.
    • 27. Qualitative and quantitative.
    • 28. ELISA can be used on most types of biological samples, such as plasma, serum, urine.
    11 May 2002
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  • 29. DISADVANTAGES OF ELISA
    • Enzyme activity may be affected by plasma constituents.
    11 May 2002
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  • 30. APPLICATIONS OF IMMUNE RESPONSE(NEUTRILIZATION AND PRECIPITATION)
    11 May 2002
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  • 31. NEUTRALIZATION
    It is a serological test used to identify used to identify toxins and antitoxins as well as viruses and viral antibodies.
    It involves antigen-antibody reaction.
    Laboratory animals are used as “indicator systems” in these tests.
    For example it is used to detect exotoxin of
    Clostridium botulinumin food.
    11 May 2002
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  • 32. NEUTRALIZATION
    Antitoxin is mixed with the food that contains toxins. Thus neutralization of toxin takes place.
    If toxin is produced by some other organism
    no neutralization occurs and mixture is lethal to animal.
    Schick test
    • The Schick test is used to determine if a person is immune to diphtheria (intradermal test)
    • 33. In this test diphtheria toxin is injected intradermally.No skin reactions occurs if person has neutralizing antibodies. A local edema occurs if no neutralizing antibodies are present , indicating person is susceptible to diphtheria.
    11 May 2002
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  • 34. Virus neutralization by specific antibodies
    11 May 2002
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  • 35. PRECIPITATION
    The immunoprecipitation technique detects soluble antigens that react with antibodies called precipitins.
    The antibodies link the antigen to form a large antibody-antigen network or lattice that settles out of solution at the equivalence zone when it becomes sufficiently large.
    In fluids,antigen and antibody are layered over each other in a thin tube.The molecules then diffuse through the fluid untill they reach equivalence zone.A visible mass of particles is now formed at interface or at bottom.
    11 May 2002
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  • 36. IMMUNODIFFUSION
    In immunodiffusion, antigens and antibodies diffuse through a semisolid gel until they reach the zone of equivalence.
    TWO METHODS
    1.Single diffusion(oudin)
    2.Double diffusion(Ouchterlony plate technique)
    • Both reactants diffuse.
    • 37. Antigen and antibody solutions are placed in wells cut into agar in petridishes.The plates are incubated and precipitation lines form at zone of equivalence.
    • 38. Used to detect fungal antigens of histoplasma,blastomyces,coccidioides.
    11 May 2002
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  • 39. 11 May 2002
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  • 40. IMMUNO-ELECTROPHORESIS
    In this technique gel electrophoresis and diffusion are combined.
    11 May 2002
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  • 41. 11 May 2002
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