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 The amount of light scatter is related to the concentration of precipitated particles (i.e., Ag/Ab complexes) Quantitative method

Precipitation Nephelometry

Double Immunodiffusion (Ouchterlony) 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

Double Immunodiffusion (Ouchterlony) 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

Double Immunodiffusion (Ouchterlony) 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

Double Immunodiffusion (Ouchterlony) 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

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

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

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

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

Western Blot Analysis

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)