Precipitation

• Aggregation of soluble antigens with antibody to form visible complex

• Directly visualize reactions

NephelometryPrecipitation

• 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

NephelometryPrecipitation

• 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

Precipitation

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

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

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

Precipitation

Double Immunodiffusion(Ouchterlony)

• No reaction• Patient’s specimen

doesn’t contain Antigen A or

• Antigen and antibody concentration not within equivalence zone

Immunofixation ElectrophoresisPrecipitation

• Proteins separated by electrophoresis

• Cellulose acetate strips impregnated with antibody applied to the gel

• Diffusion• Precipitated complexes

form

Precipitation

• Useful in demonstrating antigens in low concentrations– Urine & CSF

• Semi-quantitative

Immunofixation Electrophoresis

• 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

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)