Immunological testing Perspective 17.1 Monoclonal Antibodies • Monoclonal antibodies • Serology • Quantifying antigen – antibody reactions 17-1 17-2 Serology Quantifying antigen – antibody reactions • Antibodies • Seroconversion or rise in titer • Antibodies detect and identify antigens • Serial dilutions 17-3 17-4 Precipitation reactions • Immunodiffusion • Immunoelectrophoresis Figure 17.2 - Quantitation of immunologic tests 17-5 17-6 1 Figure 17.4 Immunodiffusion Figure 17.3 Precipitation reaction 17-7 17-8 Agglutination reactions • Direct agglutination • Indirect agglutination • Hemagglutination Figure 17.6 Immunoelectrophoresis 17-9 17-10 Direct agglutination Indirect agglutination • Cross – linking and lattice formation • Soluble antigen is coated onto particles (red bood cells, • Antibodies react with particulate antigens (red blood latex beads) cells, bacteria, fungi) • Allow for visible clumps (agglutination) • Visible clumps • Estimate amount of antibody 17-11 17-12 2 Immunofluorescence tests • Direct fluorescent antibody test • Indirect fluorescent antibody test Figure 17.7 - Agglutination reaction 17-13 17-14 Figure 17.8 - Direct and indirect fluorescent antibody test Antigen – antibody assays • Radioimmunoassay (RIA) • Enzyme – linked immunosorbant assay (ELISA) • Western blot 17-15 17-16 Enzyme – linked immunosorbant assay Radioimmunoassay (RIA) (ELISA) • Competitive inhibition assay • Color reaction assay • Measure antigen or antibody • Indirect ELISA • Ex. Measure small amounts of hormones or drugs in a • Direct ELISA clinical sample • Ex. Measure small amounts of IgE antibody (radioallergosorbent test) 17-17 17-18 3 Figure 17.9 - Indirect ELISA Figure 17.1 - Direct ELISA 17-19 17-20 Figure 17.1 Complement fixation test Western blot • Measures the binding of complement by an antigen – antibody interaction • Indicator system determine positive or negative reactions 17-21 17-22 Figure 17.1 Complement Neutralization test fixation test • Antibody bind to specific antigen (virus, toxin) • Antibody – antigen complex prevents antigen from binding (neutralization) 17-23 17-24 4 Cellular immunology test • Identification of subsets of lymphocytes • Lymphocyte response to mitogens • Cytoxic T – cell function • Cell – mediated immunity to infectious agents 17-25 5.