Immuno 10: Immunoassays/Flow Cytometry Flashcards
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
Secondary antibodies should have specificity for the ____ ____ so that the can indirectly detect the presence of an antigen.
primary antibody (of the antigen:antibody complex)
This antibody preparation will bind to any human IgG molecule. What is it?
horseradish peroxidase-conjugated anti-human IgG
A western blot analysis is a non-quantitative method of determining what?
whether an Ab sample can bind to a protein antigen
Desribe the process of running a western blot analysis.
- the antigen of interest is separated and transferred to a membrane
- the membrane is probed with antibody
- the antibody binds its specific antigen
- bound antibody is detected using a secondary antibody bound to enzyme
Is ELISA a quantitative or qualitative method of measuring antigen-specific antibody?
quantitative
What are the steps of an ELISA?
- put antigen on a plate
- add antibody; it binds antigen
- add secondary antibody with conjugated enzyme; it binds the Ab:Ag complex
- add chromogen; enzyme reacts with chromogen and ligand is visualized
The color change can be quantitated by spectrophotometry
What are the major benefits and limitations of ELISA?
benefits: very sensitive, quantitative, high throughput, cheat
limitations: requires either purified antigen or monoclonal Ab
What are the major benefits and limitations of Western blot?
benefits: purified Ag or Ab not needed; highly sensitive
limitations: non-quantitative
A serum sample from an immunized animal is commonly called ____ ____ because _____.
polyclonal antiserum because a complex mixture of antibodies can be elicited by one immunogen (antigen) since most immunogens have multiple epitopes
A flow cytometer is used to rapidly analyze cells based on what 2 features?
- physical characteristics: size and granularity–measured by light scattering
- surface marker expression–measured by fluorescence emissions when cell is conjugated to a fluorochrome
The following types of light scatter in flow cytometry are influence by what?:
- forward scatter
- side scatter
- forward scatter is influenced by size and refractive index
- the more irregular or granular a particle is, the more side scatter
A ____ ____ is a special flow cytometer that collects cells with the desired surface phenotype after they have passed through the laser.
cell sorter
Immunohistochemistry is a way to visualize tissue sections. Please describe how.
uses antibodies that are conjugated to particles that can be visualized; can see tissue sections stained this way on light or electron microscopy
Immunofluorescence is a way to visualize tissue sections. Please describe how.
uses antibodies that are conjugated to fluorochromes; visualized with a fluorescent microscope
What is an agglutination titer?
it’s the highest dilution of a serum which causes clumping of microorganisms or other particulate antigens (often RBCs)
What is a serial dilution?
repeated dilution of a sample by the same dilution factor; typically performed in a microtiter plate
What is convalescent serum? (aka convalescent-phase serum)
serum from a person who has recuperated from a particular infection, which may be of use in treating a person with the same infection; typically has low IgM and high IgG pathogen-specific antibody levels
What is acute phase serum?
serum collected from the blood of a patient that is actively infected with a pathogen; typically high IgM and low IgG pathogen-specific antibody levels; also often contains high levels of MBP, CRP, and fibrinogen
What does ELISA stand for?
Enzyme Linked Immunosorbant Assay (no this isn’t high-yield, but I know I’m going to ask myself at some point when studying later)
