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    Antigen antibody reaction mahadi ppt Antigen antibody reaction mahadi ppt Document Transcript

    • Antigen Antibody ReactionAlZaiem AlAzhari UniversityFaculty of Medical Laboratory SciencesDepartment of MicrobiologyBatch (19) Semester 4Serology and clinical ImmunologyU.Mahadi Hassan MahmoudBsc,Msc,MIBMS-Microbiologist18 April 2013 1Lecture No -1- Discuss immunoglobulin variability (ie. thevariable region) Describe bonds between the variable regionand the antigenic determinant Define antibody affinity and antibodyaviditySpecific Objectives:THE STUDENT SHOULD BE ABLE TO:
    •  Describe a precipitin curve and discuss latticeformation involving proteins verses carbohydrateantigens and be able to define "zone ofequivalence". Understand immunodiffusion in agar gels.(identity, nonidentity and partial identity) Have a conceptual understanding ofimmunoelectrophoresis, Fluorescent antibodytechniques and ELISA (enzyme-linkedimmunoassay) Define "agglutination" and understand thefunctional differences between monomeric Ab (ie.IgG) and polymeric Ab (ie. IgM and S-IgA)
    • Definitions:The "antibody affinity" of anantibody-antigen reaction is related tothe strength of attractiveness between anantibody (Fab region) and its antigenicdeterminant..The "antibody avidity" is the total strengthof binding of the Fab regions of thepopulation of antibodies evoked to anantigen, and involves the reaction with allthe antigenic determinates. Thus it is thetotal strength of the binding of antibodies toantigens.
    •  Immune Complex = Antigen-Antibody Complex [the sizedepends on the ratio of antigen toantibody].Also the student should be prepared to answer anddiscuss the following:1. List and describe the possible bonds between theimmunoglobulin variable region and an antigenic determinant.Then draw and explain a precipitin curve and "lattice formation"involving protein antigens and polyclonal Ab.2. What is meant by "hypervariable regions" onimmunoglobulins ? How do B cell clones differ in regard to thehypervariable regions of the immunoglobulins on their surface?At the level of the gene, explain what is believed to account forthese clonal diversities.3. Can two different classes of immunoglobulins have identicalvariable regions? In your answer include a discussion of theswitch mechanism.
    • ANTIGENIC DETERMINANTSINTERACT WITHSPECIFIC ANTIBODYCH2 CH3CH2 CH3IgG has a Valence of 2TWO Identical ANTIGEN BINDING SITES
    • Movementat theHingeRegionCH2 CH3CH2 CH3IgGSurfaceof anAntigeni.e.bacterialcell surfaceNon-Covalent InteractionsBall in glove fitAntigenicDeterminantVLVH
    • -Gene rearrangements andMutational Hot SpotsCharge-Charge InteractionsHydrophobic Interactions - And good fit !+-VLVH+YAntibody Affinity-+-VLVH+
    • Antigenic determinant 1Antigenic determinant 2Antigenic determinant 3Antigenic determinant 4PROTEINANTIGENYYYMUST HAVE POLYCLONAL ANTIBODYand at least two different antigenic determinantsTO CROSS-LINK PROTEIN ANTIGENSImmune Complexes
    • YYYYANTIBODYEXCESSNO CROSSLINKSNO PrecipitateYYYYY YYYExcess AntibodyYExcess Antigen = Not enoughCross-links to cause a Precipitation
    • YYMore cross-links, and higher individual affinities= higher AVIDITY of the Immune ComplexesYAb CONCANTIBODYEXCESSANTIGENEXCESSZONE ofEquivalenceNo Soluble Ag or Ab
    • Repeating Antigenic Determinantse.g. PEPTIDOGYCANCHO ANTIGENS may cross-link with MONOCLONAL AbYY YY
    • Antigen AntibodyDOUBLE DIFFUSIONImmune ComplexAntigenAntibodyAntigenImmune ComplexesZone of Equivalence
    • Rabbit Serumas antigens1:4 1:20Goat anti-rabbit serum(Antibodies to rabbit serum)Non-IdentityAntigen #1 Antigen #2
    • No Shared AntigenicDeterminantsAntigen #1 Antigen #2OUCHTERLONY ANALYSISDiffusion of Antigens and Polyclonal AntibodiesAntigen 1(Molecule #1)Antibodies to both antigensThe same Animal was injected withantigen 1 and with antigen 2Antigen 2(Molecule #2)Non-Identity
    • OUCHTERLONY ANALYSISAntigen 3is a partof antigen 4AntibodyAntigen 4Partial - IdentityRemember thatProtein Antigenshave differentantigenicdeterminantsAlso remember that thisantibody is a multi-clonal antibody such asan anti-serum to anantigenic preparationThis animalwas onlyinjected withAntigen #4OUCHTERLONY ANALYSISAntigen 3AntibodyAntigen 4Partial - Identity
    • Antigen 3Antibodiespolyclonal antibodyAntigen 4Partial - IdentityAntibodies to determinants c and d are only on Antigen 3 andthey pass by antigen 4OUCHTERLONY ANALYSISAntigen 5AntibodyAntigen 6is Antigen 5IdentityThese two Antigens are the Same MoleculeNo spikes were formedbecause:Antigenic determinants onAntigen 5 captured all theantibodies to Antigen 6 andantigenic determinants onAntigen 6 captured all theantibodies to Antigen 5
    • Antigens on Cells or on Tissue SectionsUV LightFluorescenceFluorescenceDouble layerSandwichUV LightAntigens
    • AgPeroxidase Enzyme is permanently attached to theAntibody ProbeMicrotiter ELISAAntigens are immobilized to the plastic surface of aMicrotiter PlateEnzyme Linked Immuno-Sorbant AssayELISAAgSubstrate that turnsfrom clear to greenAgPeroxidase Enzyme is permanently attached to theAntibody ProbeMicrotiter ELISAAntigens are immobilized to the plastic surface of aMicrotiter PlateEnzyme Linked Immuno-Sorbant AssayELISAAgSubstrate that turnsfrom clear to green
    • Capture ELISA -- using pre-immobilizedmouse monoclonal Ab to capture the SpecificAntigen and a second Probe monoclonalAntibody against a different antigenicdeterminantAg AgAgglutinationIgM >>IgG