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Immunological testing. Monoclonal antibodies Serology Quantifying antigen – antibody reactions. Perspective 17.1 Monoclonal Antibodies. Serology. Antibodies Antibodies detect and identify antigens. Quantifying antigen – antibody reactions. Seroconversion or rise in titer - PowerPoint PPT Presentation
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Immunological testingMonoclonal antibodiesSerologyQuantifying antigen antibody reactions
Perspective 17.1Monoclonal Antibodies
SerologyAntibodiesAntibodies detect and identify antigens
Quantifying antigen antibody reactionsSeroconversion or rise in titerSerial dilutions
Figure 17.2 - Quantitation of immunologic tests
Precipitation reactionsImmunodiffusion Immunoelectrophoresis
Figure 17.3Precipitation reactionImmune complexes large removed by phagocytes small can remain in circulation and cause disease
Figure 17.4Immunodiffusion
Figure 17.6ImmunoelectrophoresisCan be used to analyze patient Ab types
Agglutination reactionsDirect agglutinationIndirect agglutinationHemagglutination
Direct agglutinationCross linking and lattice formationAntibodies react with particulate antigens (red blood cells, bacteria, fungi)Visible clumps Estimate amount of antibody
Indirect agglutinationSoluble antigen is coated onto particles (red bood cells, latex beads)Allow for visible clumps (agglutination)
Figure 17.7 - Agglutination reactionAnti-A antibodies agglutinate; Anti-B antibodies do not.Therefore the patient has type A blood (A antigens on The surface of their red blood cells)
Immunofluorescence testsDirect fluorescent antibody testIndirect fluorescent antibody test
Figure 17.8 - Direct andindirect fluorescent antibody test
Antigen antibody assaysRadioimmunoassay (RIA)Enzyme linked immunosorbant assay (ELISA)Western blot
Radioimmunoassay (RIA)Competitive inhibition assayMeasure antigen or antibodyEx. Measure small amounts of hormones or drugs in a clinical sampleEx. Measure small amounts of IgE antibody (radioallergosorbent test)
Unlabeled Ab is used to coat wellLabeled specific Ag is added with sampleAbility of unlabeled Ag in sample to compete with labeledAg binding to Ab is measuredReduced binding indicates competition by unlabeled Ag in sampleAmount of competition a measure of unlabeled Ag levels
Enzyme linked immunosorbant assay (ELISA)Widely used; very sensitive; small volumes; little reagent; lots of samplesUsed for HIV testing of blood before it is used for transfusion
Enzyme linked immunosorbant assay (ELISA)Color reaction assayIndirect ELISADirect ELISA
Figure 17.9 - Indirect ELISA
Figure 17.1 - Direct ELISADetects human chorionic gonadotropinPresent only in pregnant women
Figure 17.1Western blotAgs separated by electorphoresisTransferred to membraneProbed with specific AbsAbs detected indirectly usinganti-HGG
Complement fixation testMeasures the binding of complement by an antigen antibody interactionIndicator system determine positive or negative reactions
Figure 17.1 Complementfixation testUsed to detect specificAbs in serum
Neutralization testAntibody bind to specific antigen (virus, toxin)Antibody antigen complex prevents antigen from binding (neutralization) Viral or toxin activity is diminished in tests
Cellular immunology testIdentification of subsets of lymphocytes (using FACS)
Cellular immunology testIdentification of subsets of lymphocytes Lymphocyte response to mitogensCytoxic T cell functionCell mediated immunity to infectious agentsAg used instead of mitogen to stimulate lymphocytes