10- Clinical Immunologic Assays-II

Question 1

Coombs Test

Answer 1

FUNCTION: Used to test if the patient is making antibody that binds to her/his own red blood cells, causing them to break up or be phagocytized.

METHOD: Patient’s erythrocytes are incubated with antibodies that react with the antibody isotype that is suspected of causing the hemolysis. If these are truly bound to the RBC, blood will aggregate and this will be a positive test.

Question 2

Immunofluorescence

Answer 2

DIRECT- light emitting compounds are linked to antibodies that directly bind antigens or indirectly bind an Ig that was previously bound to antigens in tissue

INDIRECT- patient serum containing possible antibodies is layered then washed, then anti-human Ig flourochrome is added on top of it.

Question 3

Flow Cytometry

Answer 3

Technique that can provide information on the characteristic of SINGLE CELLS IN A MIXED CELL POPULATION. The cell population to be studied is shepherded into a thin stream of single cells that have been previously incubated with the desired fluorescent-labeled monoclonal or monospecific antibody probe.

The single cell queue flows past a laser that emits a light wavelngth unique to the characteristics of the fluorochrome. If a cell has the antibody-flourochrome, the emitted wavelength is then quantified by computer software.

Question 4

Making monoclonal antibodies

Answer 4

1. Mouse spleen cells producing antibody are immunized with Antigen A.
2. Mouse spleen cells are fused with myeloma cells lacking antibody secretion via PEG.
3. Fused cell is transferred to HAT medium
4. Select hybridoma that makes antibody specific for antigen A is selected and cloned.

Question 5

Side scatter vs forward scatter

Answer 5

Seen in flow cytometry

SIDE: tell you about granulation
FORWARD: tell you about size

Question 6

Serum Protein Electrophoresis

Answer 6

Classic gel or paper electrophoresis.

After movement occurs for set time, the second step is immunofixation.

Question 7

Serum Protein Electrophoresis

Answer 7

Classic gel or paper electrophoresis.

After movement occurs for set time, the second step is immunofixation. All monoclonal antibody will migrate to a single location. All polyclonal antibody will be slightly unique in position.