P3 - Precipitation Reactions

• why user serological and molecular techniques


  • For non-culture based identification of organism
  • Direct microscopy
  • Antigen-antibody detection (serological reactions)
  • Chromatography
  • Molecular methods (e.g. PCR)
• Serological Reactions


  Antibody-antigen reactions in vitro.

  They are used in:

  1. Diagnosis of infections
  2. Epidemiological surveys
  3. Identification of antigens, e.g. enzymes

  In general for the detection and quantification of antigens and antibodies
• Titre or units


  antibody titre of a serum is the highest dilution of serum which shows an observable reaction with antigen in the particular test

  so, basically - how much of the serum can be diluted before antibodies can no longer be detected
• Sensitivity 


  Accuracy with which a test can detect the presence of infection.

  False negative is absent or minimal
• Specificity 


  Accuracy with which a test can detect the absence of infection

  False positive is absent or minimal
• Types of Serological Reactions

  Precipitation reaction

  Agglutination reaction

  Complement fixation test

  Radio-immuno assay (RIA)

  Enzyme-immuno assay (EIA)

  ELISA

  Western blot
• Precipitation 


  A soluble antigen combines with its antibody in the presence of electrolytes at an optimum temperature and pH - forming an insoluble precipitate of antigen-antibody complex

  Flocculation = precipitate suspense as floccules
• Applications of Precipitation Serological Test 


  • Slide tests = Veneral Disease Research Laboratory Test (VDRL) test for syphilis
  • Tube tests = Kahn test for syphilis
  • Immunodiffusion = Radial immunodiffusion
  • Electroimmunodiffusion = rocket electrophoresis
• Slide test, e.g. VDRL and RPR for syphilis diagnosis 


  Veneral Disease Research Laboratory Test

  Rapid Plasma Reagin Test

  ^for diagnosis of syphilis

  • a drop of each antigen and antiserum are placed on a slide and mixed = floccules appear
• Immunodiffusion (Precipitation in Gel)


  i

  • 1% soft agarose gel
  • Advantage
  • Distinct visible bands of precipitate form
  • Number of different antigens reaction mixture can be identified
• Immunodiffusion (precipitation in gel)

  • 1% soft agarose gel
  • Advantage
  • Distinct visible bands of precipitate form
  • Number of different antigens reaction mixture can be identified
  • when only antigen is diffusible = single diffusion
  • when both antigen and antibody are diffusible = double diffusion
• Radial Immunodiffusion


  • Single diffusion in two dimensions
  • Applications
  • Estimation of immunoglobulin classes in sera
  • Estimation of IgG, IgM antibodies in sera to influenza viruses
  • To determine relative concentrations of antibodies in serum
  • Estimate serum transferrin and alpha-feroprotein
  • To compare properties of two different antigens
• Ouchterlony procedure


  Double diffusion in two dimensions

  Application = Elek's test for detecting toxin of Corynebacterium diptheriae
• Properties of Corynebacterium to list if asked about in Elek's test


  • Causative agent = diphtheria toxin
  • pseudomembrane over the tonsils
  • Elek's test
  • Immunodiffusion (double diffusion) - precipitation reaction
• Zone of equivalence

  it is the concentration range where the antigen and antibody are present in sufficient quantities to form immune complexes efficiently, leading to visible reactions such as agglutination or precipitation.

  When the concentrations of antigen and antibody are too low, immune complexes may not form, resulting in false-negative reactions. Conversely, when concentrations are too high, excess antigen or antibody may saturate binding sites, hindering the formation of immune complexes and leading to false-positive reactions.