precipitation aggregation of soluble antigens with antibody to form visible complex directly...
Post on 19-Dec-2015
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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)