Immunological diagnostic tools

• Antigen-antibody complex


  Requires specific conditions (temperature, pH) to be detected, may be directly visible (agglutination) or invisible (requires probes to amplify signals)
• Methods for Ag-Ab detection

  • Precipitation
  • Agglutination
  • Hemagglutination and hemagglutination inhibition
  • Viral neutralization test
  • Radio-immunoassays
  • ELISA
  • Immunoflourescence
  • Immunoblotting
  • Immunochromatography
• Immune Complex Formation


  • Antibodies have at least 2 antigen-binding sites and most antigens have at least 2 antigenic determinants, leading to cross-linking and large aggregates
  • The extent of immune complex formation depends on the relative concentrations of antibody and antigen
• Precipitation


  Soluble antigen forms large enough immune complexes to settle out of solution
• Precipitation tests


  • VDRL slide flocculation
  • RPR card test
  • Kahn's test for syphilis
• Precipitation: Immunodiffusion


  Precipitation reaction between antibody and antigen in an agar gel medium, using single radial immunodiffusion (Mancini technique) or double diffusion agar assay (Ouchterlony technique)
• Direct agglutination


  Combination of insoluble particulate antigen with its soluble antibody, forming antigen-antibody complexes that clump/agglutinate
• Passive (indirect) agglutination


  Precipitation reaction converted into agglutination by coating antigen onto carrier particles like red blood cells, latex, gelatin, bentonite
• Agglutination tests


  • Widal test (typhoid fever)
  • Latex agglutination tests
• Hemagglutination


  Agglutination involving red blood cells
• Antibody Titer


  Determined by serial dilutions of serum containing the antibody, with the greatest dilution showing agglutination reaction
• Lateral Flow Assay


  Antigen detected by fixed antibodies on a line of chromatographic paper, with color-labeled antibodies
• Radio-immunoassays


  Radioactively labeled antibody (or antigen) competes with patient's unlabeled antibody (or antigen) for binding sites, reduction in radioactivity used to quantify patient antibody/antigen
• ELISA


  Enzyme-linked immunosorbent assay, with various types (competitive, sandwich) to detect antigens or antibodies
• Western-blot analysis


  Antigens separated by gel electrophoresis and transferred to a membrane, target proteins labeled by specific antibodies and enzyme-labeled antibodies
• Immunofluorescence


  Fluorescent dyes coupled to antibody/antigen to detect antigens or antibodies under a fluorescent microscope
• Interferon Gamma Release Assay (IGRA)

  Determines presence of helper T Lymphocytes recognizing tuberculosis-specific antigens, by measuring interferon gamma levels
• Detection of antigen denotes presence of pathogen, more important in some parasitic and fungal diseases
• Interpretation of antigen detection tests

  • Positive: Current or recent infection
  • Negative: No infection, insufficient organisms, low test sensitivity
• Interpretation of a single, acute IgM test

  • Negative: No current infection
  • Positive (Newborn): Congenital infection
  • Positive (Adult): Primary or current infection
• Interpretation of two, acute and convalescent IgG tests

  • Negative: No current infection, past infection, immunosuppression
  • Positive (4-fold rise or fall in titer): Recent infection
• Interpretation of a single IgG test

  • Negative: No exposure or immunosuppression
  • Positive (Newborn): Maternal antibodies crossed placenta
  • Positive (Adult): Evidence of infection at some undetermined time, may be significant in immunosuppression
• Elements influencing sensitivity and specificity of a test kit

  • Test format
  • Purity of antigen used
  • Type of antibody used
  • Interfering substances in sample
  • Similarity in antigenic composition of pathogens
• Monoclonal antibodies


  Antibodies produced by a clone of B lymphocyte originating from a single cell
• Antigen-antibody complex


  Requires specific conditions (temperature, pH) to be detected, may be directly visible (agglutination) or invisible (requires probes to amplify signals)
• Methods for Ag-Ab detection

  • Precipitation
  • Agglutination
  • Hemagglutination and hemagglutination inhibition
  • Viral neutralization test
  • Radio-immunoassays
  • ELISA
  • Immunoflourescence
  • Immunoblotting
  • Immunochromatography
• Immune Complex Formation


  • Antibodies have at least 2 antigen-binding sites and most antigens have at least 2 antigenic determinants, leading to cross-linking and large aggregates
  • The extent of immune complex formation depends on the relative concentrations of antibody and antigen
• Precipitation


  Soluble antigen forms large enough immune complexes to settle out of solution
• Precipitation tests


  • VDRL slide flocculation
  • RPR card test
  • Kahn's test for syphilis
• Precipitation: Immunodiffusion


  Precipitation reaction between antibody and antigen in an agar gel medium, using single radial immunodiffusion (Mancini technique) or double diffusion agar assay (Ouchterlony technique)
• Direct agglutination


  Combination of insoluble particulate antigen with its soluble antibody, forming antigen-antibody complexes that clump/agglutinate
• Passive (indirect) agglutination


  Precipitation reaction converted into agglutination by coating antigen onto carrier particles like red blood cells, latex, gelatin, bentonite
• Agglutination tests


  • Widal test (typhoid fever)
  • Latex agglutination tests
• Hemagglutination


  Agglutination involving red blood cells
• Antibody Titer


  Determined by serial dilutions of serum containing the antibody, with the greatest dilution showing agglutination reaction
• Lateral Flow Assay


  Antigen detected by fixed antibodies on a line of chromatographic paper, with color-labeled antibodies
• Radio-immunoassays


  Radioactively labeled antibody (or antigen) competes with patient's unlabeled antibody (or antigen) for binding sites, reduction in radioactivity used to quantify patient antibody/antigen
• ELISA


  Enzyme-linked immunosorbent assay, with various types (competitive, sandwich) to detect antigens or antibodies
• Western-blot analysis


  Antigens separated by gel electrophoresis and transferred to a membrane, target proteins labeled by specific antibodies and enzyme-labeled antibodies
• Immunofluorescence


  Fluorescent dyes coupled to antibody/antigen to detect antigens or antibodies, requires a fluorescent microscope