Immunodiagnosis Flashcards
LO 1. Describe the procedure used in serum protein electrophoresis and the underlying principles.
- apply serum to gel and run (size, charge)
2 stain the proteins - can and receive graph of data
How are B cells counted?
use fluorescent mAbs specific for CD19 or CD20
How are T cells counted?
use fluorescent mAbs specific for CD3, CD4, or CD8
How does flow cytometry work?
machine counts cells by shooting laser light at single-file line of them; light scatter gives info about cell size and type
How is the humoral system tested?
serum protein electrophoresis
What is one issue with electrophoresis?
it’s not very sensitive to small abnormalities
What is the order of the peaks on the serum protein electrophoresis, from left to right?
albumin (large), alpha1, alpha2, beta, gamma (tallish, wide)
Electrophoresis can be done on which body samples?
plasma, urine, CSF
What is the electrophoresis pattern look in MS?
oligoclonal peaks in the IgG region
How would you measure individual Ig subclasses or subclasses?
single radial immunodiffusion
What is the best overall test for Th1 activity?
skin test to common antigens
What is a challenge DTH test?
use dinitrofluorobenzene to sensitize T cells- 98% will become sensitized if they’re normal
Which mitogens can be used to stimulate T cells in mononuclear leukocyte preps?
PHA or Con A
What are you looking for when you use mitogens to stimulate T cells?
proliferation or IL-2, IL-4, or IFN production; tells about T cell numbers and function
What is a simple ELISA?
screen for antibodies present
What disease can a simple ELISA be used to detect?
HIV
How does a simple ELISA work?
- attach antigen to the bottom of wells on a plate.
- Add pts serum
- If Ab is present, they will stick. Wash.
- Add Ab to human IgG that has enzyme coupled to it that will change color. Wash.
- Add color wash and look for reactions.
What is a mitogen?
a plant protein that usu. binds sugar sequences on T cells
What are antinuclear antibodies?
Antibodies against autoantigens in the nucleus
How are antinuclear antibodies detected?
- Grow human cells on a slide fixed with an agent that makes the cells’ plasma membranes permeable.
- Add antibodies to penetrate to the cell’s interior.
- Add patient’s serum. Wash.
- Add fluorescein-labeled goat anti-human IgGs. Wash.
- Examine slide under UV microscope for fluorescence and pattern
What is the sandwich/capture ELISA used for?
to detect antibodies with at least 2 epitopes
How does sandwich/capture ELISA work?
add antigen to wells that contain antibody; add another antibody that will fluoresce; view resulting color
What is an example of a test that uses sandwich ELISA techniques?
rapid step test
How is RF detected?
check for agglutination of latex particles coated with IgG because RF is IgM anti-IgG
How are immune complexes detected?
put a sample in a cold environment (fridge)- if it precipitates after 1-7 days, it’s Type 3 mediated disease
What is the final product of immunohistochemistry?
black or brown enzyme product in positive test
What does a direct immunofluorescence test look for?
antigen
What does an indirect immunofluorescence test look for?
antibody
What is passive agglutination?
antigens are coupled to RBCs or latex beads; pt’s serum added; look for agglutination or not and titer.
