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Ag-Ab reactionsTests for Ag-Ab reactions
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
Affinity = attractive and repulsive forcesAffinityStrength of the reaction between a single antigenic determinant and a single Ab combining site
Calculation of AffinityAg + Ab Ag-AbApplying the Law of Mass Action:
AvidityThe overall strength of binding between an Ag with many determinants and multivalent Abs
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.
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
Factors Affecting Measurement of Ag/Ab Reactions Affinity Avidity Ag:Ab ratio Physical form of Ag
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
Agglutination TestsLattice Formation
Agglutination/HemagglutinationDefinition - tests that have as their endpoint the agglutination of a particulate antigenAgglutinin/hemagglutinin
Agglutination/HemagglutinationQuantitative agglutination testTiterProzone
Agglutination/HemagglutinationDefinition Qualitative testQuantitative test Applications Blood typing Bacterial infectionsFourfold rise in titer Practical considerations Easy Semi-quantitative
Passive Agglutination/HemagglutinationDefinition - agglutination test done with a soluble antigen coated onto a particle
ApplicationsMeasurement of antibodies to soluble antigens
Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes
Coombs (Antiglobulin)Tests Indirect Coombs TestDetects anti-erythrocyte antibodies in serum
Coombs (Antiglobulin)Tests ApplicationsDetection of anti-Rh AbAutoimmune hemolytic anemia
Agglutination/Hemagglutination InhibitionDefinition - test based on the inhibition of agglutination due to competition with a soluble Ag
Agglutination/Hemagglutination InhibitionApplicationsMeasurement of soluble AgPractical considerationsSame as agglutination test Definition
Precipitation TestsLattice Formation
Radial Immunodiffusion (Mancini)InterpretationDiameter of ring is proportional to the concentrationQuantitativeIg levels Method Ab in gel Ag in a well
ImmunoelectrophoresisMethodAgs are separated by electrophoresis Interpretation Precipitin arc represent individual antigensAb is placed in trough cut in the agar
ImmunoelectrophoresisMethodInterpretationQualitativeRelative concentration
Countercurrent electrophoresisMethodAg and Ab migrate toward each other by electrophoresisUsed only when Ag and Ab have opposite charges QualitativeRapid
Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA)Lattice formation not required
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
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
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
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
Tests for Cell Associated AntigensLattice formation not required
Immunofluorescence Direct Ab to tissue Ag is labeled with fluorochrome
ImmunofluorescenceIndirectAb to tissue Ag is unlabeledFluorochrome-labeled anti-Ig is used to detect binding of the first Ab.Qualitative to Semi-Quantitative
Immunofluorescence Flow Cytometry Cells in suspension are labeld with fluorescent tag Direct or Indirect Fluorescence Cells analyzed on a flow cytometer
Immunofluorescence Flow Cytometry cont. Data displayedGreen Fluorescence IntensityNumber of CellsUnstained cellsFITC-labeled cellsOne Parameter Histogram
Assays Based on ComplementLattice formation not required
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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