Immuno 10: Immunoassays/Flow Cytometry Flashcards
(43 cards)
What are you looking at when you see a precipitin line in the agar gel of an immunoassay?
a precipitate where the antigen has bound and cross-linked the antigen; indicates that yes, the antibody has specificity for that antigen
Immuno-double diffusion is a technique for determining what about a given antibody and antigen?
whether the anitbody has specificity for that antigen
True or false: Immuno-double diffusion can be used to analyze a complex array of antigens.
False - can be used with simple antigen mixtures only
True or false: Immunoelectrophoresis can be used to analyze a complex array of antigens.
True
Is immunoelectrophoresis a qualitative or quantitative assay?
qualitative
The immunoelectrophoresis set-up includes an agar gel with a central ____ and a single ____ on each side.
central trough; single well
What is added to the central trough of an immunoelectrophoresis assay, and what is added to the flanking wells?
antibodies added to trough; antigens added to wells
Hemagglutination can be used to ____ and ____ antibodies that are specific for a particular antigen.
detect and measure
How does the hemagglutination assay measure antibodies?
antibody sample is taken and mixed with RBCs that have been prepared to express the antigen; when a sufficient amount of antibody binds antigen, the RBCs agglutinate (cross-link) and sink as a mat to the bottom of the well; if insufficient the RBCs will fall to form a red pellet
What’s the difference between a Direct Coomb’s test and an Indirect Coomb’s test?
Direct Coomb’s - detects Abs/complement factors already bound to patient’s RBCs in vivo
Indirect Coomb’s - detects Abs in patient’s serum that may bind to RBCs when introduced
A positive direct Coomb’s test is indicative of what?
that the patient has an autoimmune disease where self-reactive antibodies/complement factors bind and destroy self RBCs
A positive indirect Coomb’s test is indicative of what?
that the patient has antibodies in circulation that–while they aren’t self-reactive to the patient–may bind to human RBCs in the case of a transfusion, or to fetal RBCs in pregnancy
True or false: a patient needs a positive indirect Coomb’s test to receive a transfusion.
False - a patient needs a negative indirect Coomb’s test before receiving a transfusion; cross-matching of blood prior to transfusion is critical
What hemagglutination test is diagnostic for Epstein-Barr virus?
Monospot test, aka Paul Brunnel test
How is the monospot test similar to the indirect Coomb’s test, and how is it different?
similar in the process, different in that it uses sheep blood instead of human to test agglutination
What variation on the monospot test does the Paul-Brunnell-Davidsohn test carry?
it differentiates among the three types of heterophile sheep erythrocyte agglutinins:
- those associated with infectious mononucleosis
- those associated with serum sickness
- natural antibodies against Forssman antigen.
Are hemagglutination assays sensitive?
Yes, fairly: they can detect antibodies at less than 1µg/ml
What are the steps of the complement fixation assay? (briefly described)
- antigen added to well with serum containing Abs
- complement added; if Ag:Ab complexes are present (meaning Ag-specific Ab present in serum) they will fix and consume the complement
- add sRBC (indicator cells); if there’s any free complement it will form MAC and lyse the cells
SO: if there are RBCs that agglutinate, it will be because they weren’t lysed by complement because it was fixed by the immune complexes.
–> sRBCs present = Ab in the serum
A complement fixation assay is used clinically to identify the presence of ____ specific for ________.
antibodies; a variety of common human pathogens
Affinity chromatography is a method of ____ ____.
purifying antibodies
Affinity chromatography takes advantage of ____’s affinity for binding the Fc region of ____, ____, and ____ in order to purify these antibodies.
protein A; IgG1, IgG2, and IgG4
In affinity chromatography, the molecule that will be eluting the antibodies is ____ bounded to sepharose beads which are placed in an affinity column.
covalently
What are some ways in which antibodies can be covalently modified in order to be more easily detected in assays?
- labeled with radionucleotides
- labeled with enzymes (for color reaction)
- coupled to fluorochromes
- labeled with proteins that can be used as amplification system
Antibody conjugates are those that have been labeled with ___________________.
Either a radionucleotide, enzyme, fluorochrome, or with a component of an amplification system
