117-1Immunological testing• Monoclonal antibodies• Serology• Quantifying antigen – antibody reactions17-2Perspective 17.1Monoclonal Antibodies217-3Serology• Antibodies• Antibodies detect and identify antigens17-4Quantifying antigen – antibody reactions• Seroconversion or rise in titer• Serial dilutions317-5Figure 17.2 - Quantitation of immunologic tests17-6Precipitation reactions• Immunodiffusion• Immunoelectrophoresis417-7Figure 17.3Precipitation reactionImmune complexeslarge removed by phagocytessmall can remain in circulationand cause disease17-8Figure 17.4Immunodiffusion517-9Figure 17.6ImmunoelectrophoresisCan be used to analyzepatient Ab types17-10Agglutination reactions• Direct agglutination• Indirect agglutination• Hemagglutination617-11Direct agglutination• Cross – linking and lattice formation• Antibodies react with particulate antigens (red blood cells, bacteria, fungi)• Visible clumps • Estimate amount of antibody17-12Indirect agglutination• Soluble antigen is coated onto particles (red bood cells, latex beads)• Allow for visible clumps (agglutination)717-13Figure 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)17-14Immunofluorescence tests• Direct fluorescent antibody test• Indirect fluorescent antibody test817-15Figure 17.8 - Direct andindirect fluorescent antibody test17-16Antigen – antibody assays• Radioimmunoassay (RIA)• Enzyme – linked immunosorbant assay (ELISA)• Western blot917-17Radioimmunoassay (RIA)• Competitive inhibition assay• Measure antigen or antibody• Ex. Measure small amounts of hormones or drugs in a clinical sample• Ex. 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 levels17-18Enzyme – 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 transfusion1017-19Enzyme – linked immunosorbant assay (ELISA)• Color reaction assay• Indirect ELISA• Direct ELISA17-20Figure 17.9 - Indirect ELISA1117-21Figure 17.1 - Direct ELISADetects human chorionic gonadotropinPresent only in pregnant women17-22Figure 17.1Western blotAgs separated by electorphoresisTransferred to membraneProbed with specific AbsAbs detected indirectly usinganti-HGG1217-23Complement fixation test• Measures the binding of complement by an antigen –antibody interaction• Indicator system determine positive or negative reactions17-24Figure 17.1Complementfixation testUsed to detect specificAbs in serum1317-25Neutralization test• Antibody bind to specific antigen (virus, toxin)• Antibody – antigen complex prevents antigen from binding (neutralization) • Viral or toxin activity is diminished in tests17-26Cellular immunology test• Identification of subsets of lymphocytes (using FACS)1417-27Cellular immunology test• Identification of subsets of lymphocytes Lymphocyte response to mitogens• Cytoxic T – cell function• Cell – mediated immunity to infectious agentsAg used instead of mitogen to stimulate