23 Page Flashcards
Radial Immunodiffusion?
antibodies are uniformly distributed in a support gel, and antigens are applied to a well cut into the gel.
What happens as the antigen diffuses out from the well in Radial Immunodiffusion?
Antigen-antibody combination occurs in changing proportions until the zone of equivalence is reached, forming a stable lattice network in the gel.
How is antigen concentration determined in Radial Immunodiffusion?
The area of the ring obtained is proportional to the antigen concentration, compared with a standard curve using antigens of known concentration.
What is the END POINT in the Mancini method?
The antigen is allowed to diffuse to completion, and when equivalence is reached, there is no further change in ring diameter.
MANCINI
ENDPOINT
Fahey and McKelvey
KINETIC
What is the reading time for IgG in the Mancini method?
24 hours.
What is the KINETIC method in Fahey and McKelvey?
Measurements are taken before the point of equivalence is reached, and the antigen is not allowed to diffuse completely.
What is the reading time for IgM in the KINETIC method?
50-72 hours (2-3 days).
How is the antigen concentration calculated in Radial Immunodiffusion?
d2=Ag conc.
The square of the diameter is directly proportional to the concentration of the antigen.
What type of graph is plotted in Mancini method?
A graph is plotted with concentrations of standards on the x-axis versus the diameter squared on the y-axis.
What is Ouchterlony Double Diffusion?
A qualitative gel precipitation technique where both antigen and antibody diffuse from wells cut in the gel.
What does the pattern obtained in Ouchterlony Double Diffusion indicate?
Whether or not the antigens are identical.
How are the wells arranged in Ouchterlony Double Diffusion?
Plates are set up with a central well surrounded by four to six equidistant outer wells.
What is placed in the central well of Ouchterlony Double Diffusion?
A multispecific antibody.
What is the incubation period for Ouchterlony Double Diffusion?
Between 12-48 hours in a moist chamber.
What do precipitin lines in Ouchterlony Double Diffusion indicate?
They form where the moving front of antigen meets that of antibody at the point of equivalence.
What are some uses of Ouchterlony Double Diffusion?
To identify fungi such as Aspergillus, Blastomyces, Coccidioides, and Candida.
Serological Identity/ Line of identity/ Line of fusion in Ouchterlony Double Diffusion?
The two antigens are identical.
Line of partial intersection/ Partial identity indicate?
The antigens share a common epitope.
Line of non-identity/Line of intersection indicate?
The antigens share no identical determinants/not the same
Formation of smooth curve/arc
Serological identity
Partial identity
Formation of spur
What is Rocket Immunoelectrophoresis?
A combination of radial immunodiffusion (RID) and electrophoresis.
Developed by Laurell.
Distribution in Rocket Immunoelectrophoresis?
The antibody is distributed in the gel, and the height of the rocket corresponds to the amount of antigen in the sample.
height of the rocket
equals to amount of Ag in sample
rocket immunoelectrophoresis is developed by
Laurell
one-dimension electroimmunodiffusion
rocket immunoelectrophoresis
What is the arrangement of samples in Rocket Immunoelectrophoresis?
Standards are in wells 1 to 3
Patient samples are in wells 4 to 6.
Well 4-no Ag, no ring is formed
Well 5-low conc.
Well 6-high conc.
Well 4 contains no antigen, Well 5 has a low concentration of antigen, and Well 6 has a high concentration of antigen.
Immunoelectrophoresis?
combines protein electrophoresis and immunodiffusion.
How is the antigen and antibody arranged in Immunoelectrophoresis?
The antigen is placed in a well and electrophoresed, while the antibody is placed in a trough parallel to the separated proteins.
What does an abnormal contour of the precipitin arc indicate?
monoclonal gammopathy.
What is the purpose of Serum IEP?
For the detection of monoclonal gammopathy.
Urine IEP?
Bence Jones protein.
What is Immunofixation Electrophoresis?
A technique that combines protein electrophoresis and immunoprecipitation.
How is the specimen processed in Immunofixation Electrophoresis?
The specimen is electrophoresed at 6 positions, and monospecific antisera is layered on each position.
What types of antibodies are included in Immunofixation Electrophoresis (monospecific antisera)?
Antibodies to gamma, alpha, mu (heavy chains), and kappa, lambda (light chains).
GAM
KL
What is the outcome of Immunofixation Electrophoresis?
Ag-Ab complexes form, are washed, and stained to visualize the results.
What is the use of Immunofixation Electrophoresis?
Used to identify heavy and light chains involved in monoclonal gammopathies.
