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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