ag-ab reactions 2001
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Ag-Ab reactionsTests for Ag-Ab reactions
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Nature of Ag/Ab Reactions Lock and Key Concept Non-covalent Bonds Hydrogen bonds Electrostatic bonds Van der Waal forces Hydrophobic bonds Reversible Multiple Bonds
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Affinity = attractive and repulsive forcesAffinityStrength of the reaction between a single antigenic determinant and a single Ab combining site
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Calculation of AffinityAg + Ab Ag-AbApplying the Law of Mass Action:
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AvidityThe overall strength of binding between an Ag with many determinants and multivalent Abs
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SpecificityThe ability of an individual antibody combining site to react with only one antigenic determinant.The ability of a population of antibody molecules to react with only one antigen.
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Cross ReactivityThe ability of an individual Ab combining site to react with more than one antigenic determinant.The ability of a population of Ab molecules to react with more than one Ag
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Factors Affecting Measurement of Ag/Ab Reactions Affinity Avidity Ag:Ab ratio Physical form of Ag
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Tests Based on Ag/Ab ReactionsAll tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexesAll tests based on Ag/Ab reactions can be used to detect either Ag or Ab
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Agglutination TestsLattice Formation
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Agglutination/HemagglutinationDefinition - tests that have as their endpoint the agglutination of a particulate antigenAgglutinin/hemagglutinin
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Agglutination/HemagglutinationQuantitative agglutination testTiterProzone
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Agglutination/HemagglutinationDefinition Qualitative testQuantitative test Applications Blood typing Bacterial infectionsFourfold rise in titer Practical considerations Easy Semi-quantitative
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Passive Agglutination/HemagglutinationDefinition - agglutination test done with a soluble antigen coated onto a particle
ApplicationsMeasurement of antibodies to soluble antigens
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Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes
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Coombs (Antiglobulin)Tests Indirect Coombs TestDetects anti-erythrocyte antibodies in serum
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Coombs (Antiglobulin)Tests ApplicationsDetection of anti-Rh AbAutoimmune hemolytic anemia
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Agglutination/Hemagglutination InhibitionDefinition - test based on the inhibition of agglutination due to competition with a soluble Ag
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Agglutination/Hemagglutination InhibitionApplicationsMeasurement of soluble AgPractical considerationsSame as agglutination test Definition
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Precipitation TestsLattice Formation
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Radial Immunodiffusion (Mancini)InterpretationDiameter of ring is proportional to the concentrationQuantitativeIg levels Method Ab in gel Ag in a well
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ImmunoelectrophoresisMethodAgs are separated by electrophoresis Interpretation Precipitin arc represent individual antigensAb is placed in trough cut in the agar
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ImmunoelectrophoresisMethodInterpretationQualitativeRelative concentration
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Countercurrent electrophoresisMethodAg and Ab migrate toward each other by electrophoresisUsed only when Ag and Ab have opposite charges QualitativeRapid
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Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA)Lattice formation not required
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Competitive RIA/ELISA for Ag MethodDetermine amount of Ab needed to bind to a known amount of labeled Ag
Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
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Competitive RIA/ELISA for Ag Method cont.Determine amount of labeled Ag bound to Ab NH4SO4 anti-Ig Immobilize the Ab Quantitative Most sensitive test
Concentration determined from a standard curve using known amounts of unlabeled Ag
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Solid Phase Non-Competitive RIA/ELISAAb detectionImmobilize AgIncubate with sampleAdd labeled anti-IgAmount of labeled Ab bound is proportional to amount of Ab in the sample Quantitative
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Solid Phase Non-Competitive RIA/ELISAAg detectionImmobilize AbIncubate with sampleAdd labeled antibodyAmount of labeled Ab bound is proportional to the amount of Ag in the sample Quantitative
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Tests for Cell Associated AntigensLattice formation not required
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Immunofluorescence Direct Ab to tissue Ag is labeled with fluorochrome
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ImmunofluorescenceIndirectAb to tissue Ag is unlabeledFluorochrome-labeled anti-Ig is used to detect binding of the first Ab.Qualitative to Semi-Quantitative
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Immunofluorescence Flow Cytometry Cells in suspension are labeld with fluorescent tag Direct or Indirect Fluorescence Cells analyzed on a flow cytometer
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Immunofluorescence Flow Cytometry cont. Data displayedGreen Fluorescence IntensityNumber of CellsUnstained cellsFITC-labeled cellsOne Parameter Histogram
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Assays Based on ComplementLattice formation not required
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Complement FixationAg mixed with test serum to be assayed for AbStandard amount of complement is addedErythrocytes coated with Abs is addedAmount of erythrocyte lysis is determinedAg AgMethodology
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