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Precipitation

Precipitation. Aggregation of soluble antigens with antibody to form visible complex Directly visualize reactions. Precipitation. Nephelometry. Measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension

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Precipitation

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  1. Precipitation • Aggregation of soluble antigens with antibody to form visible complex • Directly visualize reactions

  2. Precipitation Nephelometry • Measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension • The amount of light scatter is related to the concentration of precipitated particles (i.e., Ag/Ab complexes) • Quantitative method

  3. Precipitation Nephelometry

  4. Precipitation Double Immunodiffusion(Ouchterlony) • Antibody and antigen diffuse • Visible precipitate will form if: • Ab recognizes and has an affinity for antigen • Ag/Ab concentration within equivalence zone • All other physical conditions conducive for Ag/Ab reaction (i.e., pH, temp, etc.) • Rxn may take hours to days

  5. Precipitation Double Immunodiffusion(Ouchterlony) • Line of identity • Patient’s serum has an antibody to antigen A • The antibody is identical to the known antibody to antigen A

  6. Precipitation Double Immunodiffusion(Ouchterlony) • Line of partial-identity • Patient’s serum contains an antigen that reacts with anti-Aa • Patient’s antigen similar but not identical to the known antigen

  7. Precipitation Double Immunodiffusion(Ouchterlony) • Line of non-identity • Patient’s serum contains an antibody to the antigen AB • Patient’s antibody different than the known antibody

  8. Precipitation Double Immunodiffusion(Ouchterlony) • No reaction • Patient’s specimen doesn’t contain Antigen A or • Antigen and antibody concentration not within equivalence zone

  9. Precipitation Immunofixation Electrophoresis • Proteins separated by electrophoresis • Cellulose acetate strips impregnated with antibody applied to the gel • Diffusion • Precipitated complexes form

  10. Precipitation Immunofixation Electrophoresis • Useful in demonstrating antigens in low concentrations • Urine & CSF • Semi-quantitative

  11. Western Blot Analysis • Some references consider • a type of precipitation rxn (IFE) • “conceptually similar” to a precipitation rxn • Proteins (i.e., antigens of virus) separated in gel via electrophoresis • Separated proteins transferred or “blotted” onto nitrocellulose paper • Incubate with serum to detect antibodies to separated proteins

  12. Western Blot Analysis

  13. Flocculation • Soluble antigen and antibody form precipitate of fine particles • Visible (microscopically or macroscopically) because lattice is forced to remain in a confined space • Syphilis testing (RPR & VDRL)

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