Precipitation Methods Flashcards
Principle: Soluble antigen combines with soluble antibody to produce visible insoluble complexes
Precipitation
Clumping together of particles to form visible masses over a narrow range of antigen concentration
Flocculation
Principle: Soluble antigens react with specific antibodies to form a precipitate of fine particles
Flocculation
Applications of Flocculation
Venereal Disease Research Laboratory (VDRL) tests
Rapid Plasma Reagin (RPR)
Principle: Light scattering by immune complexes is measured
● scattering of light is proportional to the size and amount of immune complexes formed
Nephelometry
Applications of Nephelometry
Immunoglobulins
Complement
C-reactive protein
Measures the decrease in light intensity in a solution containing immune complexes
Turbidimetry
Measures the reduction of light transmitted at 180 ° angle.
Turbidimetry
Measures transmitted light at 90 ° angle
Nephelometry
if only one reactant (usually antigen) is moving
Single Diffusion
if both antigen and antibody are moving through the medium
Double diffusion
if the reaction in a medium have only one effective dimension for antigen and antibody migration (i.e., up and down)
Single dimension
if the reaction is in circular holes (i.e., wells) cut in a gel on a flat surface, diffuses from the wells radially
Double dimension
Principle: Known Antibody fixed in agar + Unknown Antigen (overlaid) → Precipitin lines
Single Linear Diffusion (SLD) or Oudin Technique
Applications of Single Linear Diffusion (SLD) or Oudin Technique
Detects multiple antigen- antibody reactions
Principle: Known Antibody fixed in agar + Unknown Antigen (well cut in agar plate) → Precipitin ring
Single Radial diffusion
2 types of Single Radial diffusion
Fahey Method & Mancini Method
diameter of precipitin ring at 24 hours (Read before it reaches the maximum at 6-12 hours)
Fahey Method
area of precipitin ring formed at 48 hours.
Mancini Method
Uses antibody not in excess.
Fahey method
● Diffusion time for result reading is critical.
● Diffusion endpoints may not be reached,
affecting accuracy.
Fahey method
Uses excess antibody in the gel for higher
sensitivity and accuracy.
Mancini method
More commonly used in commercial
immunodiffusion plates.
Mancini method
Principle: Antigen diffuses out of well in a gel containing antibody → Precipitin ring forms → Diameter proportional to the concentration of antigen
Radial Immunodiffusion (RID)
technique used to measure antigen concentration based on the formation of a lattice network between ag and ab in a gel.
Radial Immunodiffusion (RID)
2 types of Radial Immunodiffusion (RID)
Endpoint Method (Mancini Method)
Kinetic Method (Fahey Method)
● Ring diameter readings are taken at about 19 hours before equivalence is reached.
● The diameter is proportional to the log of the concentration.
Kinetic Method (Fahey Method)
● Antigen is allowed to diffuse to completion until equivalence is reached, indicated by no further change in ring diameter.
● The square of the diameter is directly proportional to the antigen concentration.
Endpoint Method (Mancini Method)
Applications of Radial Immunodiffusion (RID)
Immunoglobulins, complement
No longer commonly performed except for low-volume testing of IgD and IgG
Radial Immunodiffusion (RID)
Principle: Antigens and antibodies diffuse out from wells cut in gel and precipitin lines where they meet
Ouchterlony technique / double immunodiffusion
Ouchterlony technique / double immunodiffusion
Three basic reaction patterns:
Identity
Non-identity
Partial identity
a single smooth arc of precipitation forms between the antigens and antibodies
Identity
two separate lines of precipitation cross each other
Non-identity
two precipitating lines meet, forming a
spur
Partial identity
Applications of Ouchterlony technique / double immunodiffusion
Fungal antigens
Extractable nuclear antigens
Principle: Antigens and antibodies are placed in wells that are directly opposite one another in a gel → an electrophoretic charge is applied to drive the reactants toward each other → precipitin band forms where they meet
Countercurrent immunoelectroph oresis (CIE)
● To rapidly check any antisera for the presence and specificity of antibodies for a particular antigen.
● To detect antigens and/or antibodies in serum for diagnosis of a particular disease
Countercurrent immunoelectroph oresis (CIE)
Applications of Countercurrent immunoelectroph oresis (CIE)
Bacterial antigens
Principle: Proteins are separated by electrophoresis, then subjected to double diffusion with reagent antibodies placed in a trough cut in the agar → shape, intensity, and location of the precipitin arcs developed are compared with those of a normal control
Immunoelectrophoresis (IEP)
Applications of Immunoelectrop horesis (IEP)
Serum proteins, including immunoglobulins
Principle: Proteins are separated by electrophoresis → cellulose acetate strip impregnated with antiserum is placed on the separated proteins → the antiserum diffuses into the gel, and antigen-antibody complexes precipitate
Immunofixation electrophoresis (IFE)
Gel diffusion + Electrophoresis
Immunoelectrop horesis (IEP)
Protein electrophoresis + immunoprecipitation
Immunofixation electrophoresis (IFE)
Applications of Immunofixation electrophoresis (IFE)
Identification of immunoglobulin s in monoclonal gammopathies, Bence-Jones proteins
Principle: An electrical charge is applied to a RID assay → the height of the rocket-shaped precipitin band is proportional to the concentration of antigen
Rocket Electrophoresis
Applications of Rocket Electrophoresis
Immunoglobulins, complement, alpha-fetoprotein
