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Classification of antigen-antibody interactions:. - Primary serological tests: (Marker techniques) e.g. Enzyme linked immuonosorbent assay (ELISA) Immuno flurescent antibody technique (IFAT) Radio immuno assay (RIA) - Secondary serological tests: e.g. Agglutination tests
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Classification of antigen-antibody interactions: • - Primary serological tests: (Marker techniques) e.g. • Enzyme linked immuonosorbent assay (ELISA) • Immuno flurescent antibody technique (IFAT) • Radio immuno assay (RIA) • - Secondary serological tests: e.g. • Agglutination tests • Complement fixation tests (CFT) • Precipitation tests • Serum neutralization tests (SNT) • Toxin-antitoxin test
Agglutination : • - Antigens are whole cells (eg. RBC or bacteria). • - Antibodies cross-link the antigens. • - Types of tests : • - Rapid plasma test (syphilis). • - Cold agglutinin test (mycoplasma). • - Weil-Felix test (rickettsial). • - Latex agglutination test (pregnancy).
Agglutination : ( continued ) - Agglutination reactions involve whole cell antigens, while precipitation reactions involve soluble antigens. - Cellular/molecular view of agglutination and Precipitation reactions that produce visible Ag-Ab complexes.
Rapid plasma reagin ( RPR test ) - The rapid plasma reagin (RPR) test is available as a kit. The patient's serum is mixed with cardiolipin on a plastic-coated card that can be examined with the naked eye.
Precipitation • - Soluble antigens. • - Binding of antibody to antigen, makes the complex insoluble or visible. • - Types of test : • - Tube precipitation. • - Ouchterlony (double diffusion). • - Immunoelectrophoresis.
Precipitation : ( Continued ) - Precipitation reactions
Precipitation : ( Continued ) Ouchterlony (double diffusion)
Precipitation : ( Continued ) Precipitation curve :
Complement fixation • - Antibody (lysin), antigen, complement, and sensitized sheep rbc are required. • - Complement is fixed to a Ab-Ag-complex. • -Fixed complement cannot participate in rbc lysis positive reaction or identification.
Nephelometry (Turbidimetry ) - Nephelometry is a technique used in immunology to determine levels of IgM, IgG, and IgA.
Immunofluorescence • - A monoclonal antibody labeled with a fluorescent dye : • - Direct testing. • - Indirect testing.
Immunofluorescence testing - An example of direct and indirect immunofluorescence testing.
Common assays - Radioimmunoassay (RIA). - Enzyme-Linked Immunosorbent Assay (ELISA).
Radioimmunoassay (RIA) • - Antibody or antigen is labeled. • - Detect corresponding antibody or antigen. • - Competition between unlabeled and labeled molecule.
Enzyme-Linked Immunosorbent Assay (ELISA) • - Safer & less costly than RIAs. - ELISA methods can detect any infectious disease provided if we have antibodies and antigen to any infection, enzyme or any substance.
Enzyme-Linked Immunosorbent Assay (ELISA) • - Horseradish peroxidase or alkaline phosphatase. • - Enzyme releases dye (chromogen). • - Types of test : • - Indirect ( Non-competitive ). • - Capture or Sandwich. • - Competitive ELISA.
Ex: detecting the presence of serum Abs against HIV An example of the indirect ELISA method