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