Diagnosing the Immune System Flashcards
Explain serum protein electrophoresis
test the humoral arm of the immune system.
- Apply serum, turn on voltage, run
- Stain proteins
- Scan to get a nice graph.
Explain normal pattern in serum protein electrophoresis
Normal distribution, left to right: Albumin, alpha1, alpha2, beta, gamma. Look at pic in lecture.
Explain serum protein electrophoresis pattern seen in pts with selective IgA deficiency.
Normal. it could not pick up selective IgA deficiency, because IgA runs pretty much together with the much larger IgG (gamma) band.
Explain serum protein electrophoresis pattern seen in pts with multiple myeloma.
Sharp spike in Gamma band
Explain serum protein electrophoresis pattern seen in pts with severe pyogenic infections.
Broad elevation in the gamma band.
Explain serum protein electrophoresis pattern seen in pts with hypogammaglobinemia
Ver small or no gamma band.
Describe single radical immunodiffusion.
measure levels of individual immunoglobulin classes or subclasses using the concept of immunodiffusion. Immunodiffusion can be used to measure any other multivalent antigen (one that can form a precipitate with an appropriate antibody)
How do you find out if a patient has antibody to a soluble or particulate antigen?
Simple ELISA
Indirect vs direct immunofluorescence techniques.
Direct immunofluorescence: A throat swab is smeared on a slide, and fluorescent-labelled antibodies to known bacterial antigens are poured over it. A test for antigen.
Indirect immunofluorescence: Known bacteria are placed on a slide, and the patient’s serum
is poured over. After washing, fluorescent- labelled goat anti-human Ig is poured over.
A test for antibody.
Passive agglutination vs precipitation`
Passive agglutination: we couple small Ags to red blood cells or latex beads, and add dilutions of the patient’s serum, looking for an agglutination titer (titer is the reciprocal of the highest dilution that will still do something)
-Precipitation: but hopelessly insensitive, and never done for diagnosis.
Describe the principle of ELISA test used to measure antigen.
-Capture ELISA (Antigen): Divalent antigen must be used. You start by making or buying
two monoclonal antibodies to the antigen,
each to a different epitope. Put one mAb on a
plate so that it’s stuck there. That’s the
capture antibody. Add the patient’s serum.
Wash off anything that isn’t bound. Then add
the second antibody, which will stick to the other epitope on the antigen in proportion to how much antigen’s there. The second antibody has an enzyme (peroxidase). Now add peroxidase substrate and a color change takes place. Finally, measure the intensity of the product color in a plate spectrophotometer, also called an ELISA reader.
Describe the principle of ELISA test used to measure Ab.
-Simple ELISA (Antibody): As is done in the HIV antibody screen, antigen is coupled to a plate, then the test serum is added; if there is antibody to the antigen it will bind. It is then identified using an enzyme-coupled antibody to the specific class of the expected serum antibody.
What is the best test to evaluate T cell immunocompetence?
skin test with common antigens
Describe tests to evaluate T cell number and function in the lab.
Killer cell assays
Describe flow cytometry
they take cells in suspension and pump them through an orifice so small that the cells emerge in single file in a very fine stream. Lasers illuminate the cells and light emitted or scattered by each cell is collected by photomultipliers connected to a fast computer system. Light scatter gives information about cell size and cytoplasmic granularity, and if the cell has bound a fluorescent-tagged antibody, the fluorescent light emitted is quantified
