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CMOD- Block1 > Immuno 10: Immunoassays/Flow Cytometry > Flashcards

Immuno 10: Immunoassays/Flow Cytometry Flashcards

1
Q

What are you looking at when you see a precipitin line in the agar gel of an immunoassay?

A

a precipitate where the antigen has bound and cross-linked the antigen; indicates that yes, the antibody has specificity for that antigen

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2
Q

Immuno-double diffusion is a technique for determining what about a given antibody and antigen?

A

whether the anitbody has specificity for that antigen

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3
Q

True or false: Immuno-double diffusion can be used to analyze a complex array of antigens.

A

False - can be used with simple antigen mixtures only

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4
Q

True or false: Immunoelectrophoresis can be used to analyze a complex array of antigens.

A

True

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5
Q

Is immunoelectrophoresis a qualitative or quantitative assay?

A

qualitative

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6
Q

The immunoelectrophoresis set-up includes an agar gel with a central ____ and a single ____ on each side.

A

central trough; single well

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7
Q

What is added to the central trough of an immunoelectrophoresis assay, and what is added to the flanking wells?

A

antibodies added to trough; antigens added to wells

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8
Q

Hemagglutination can be used to ____ and ____ antibodies that are specific for a particular antigen.

A

detect and measure

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9
Q

How does the hemagglutination assay measure antibodies?

A

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

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10
Q

What’s the difference between a Direct Coomb’s test and an Indirect Coomb’s test?

A

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

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11
Q

A positive direct Coomb’s test is indicative of what?

A

that the patient has an autoimmune disease where self-reactive antibodies/complement factors bind and destroy self RBCs

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12
Q

A positive indirect Coomb’s test is indicative of what?

A

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

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13
Q

True or false: a patient needs a positive indirect Coomb’s test to receive a transfusion.

A

False - a patient needs a negative indirect Coomb’s test before receiving a transfusion; cross-matching of blood prior to transfusion is critical

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14
Q

What hemagglutination test is diagnostic for Epstein-Barr virus?

A

Monospot test, aka Paul Brunnel test

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15
Q

How is the monospot test similar to the indirect Coomb’s test, and how is it different?

A

similar in the process, different in that it uses sheep blood instead of human to test agglutination

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16
Q

What variation on the monospot test does the Paul-Brunnell-Davidsohn test carry?

A

it differentiates among the three types of heterophile sheep erythrocyte agglutinins:

  1. those associated with infectious mononucleosis
  2. those associated with serum sickness
  3. natural antibodies against Forssman antigen.
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17
Q

Are hemagglutination assays sensitive?

A

Yes, fairly: they can detect antibodies at less than 1µg/ml

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18
Q

What are the steps of the complement fixation assay? (briefly described)

A
  1. antigen added to well with serum containing Abs
  2. complement added; if Ag:Ab complexes are present (meaning Ag-specific Ab present in serum) they will fix and consume the complement
  3. 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
19
Q

A complement fixation assay is used clinically to identify the presence of ____ specific for ________.

A

antibodies; a variety of common human pathogens

20
Q

Affinity chromatography is a method of ____ ____.

A

purifying antibodies

21
Q

Affinity chromatography takes advantage of ____’s affinity for binding the Fc region of ____, ____, and ____ in order to purify these antibodies.

A

protein A; IgG1, IgG2, and IgG4

22
Q

In affinity chromatography, the molecule that will be eluting the antibodies is ____ bounded to sepharose beads which are placed in an affinity column.

A

covalently

23
Q

What are some ways in which antibodies can be covalently modified in order to be more easily detected in assays?

A
  • labeled with radionucleotides
  • labeled with enzymes (for color reaction)
  • coupled to fluorochromes
  • labeled with proteins that can be used as amplification system
24
Q

Antibody conjugates are those that have been labeled with ___________________.

A

Either a radionucleotide, enzyme, fluorochrome, or with a component of an amplification system

25
Q

Secondary antibodies should have specificity for the ____ ____ so that the can indirectly detect the presence of an antigen.

A

primary antibody (of the antigen:antibody complex)

26
Q

This antibody preparation will bind to any human IgG molecule. What is it?

A

horseradish peroxidase-conjugated anti-human IgG

27
Q

A western blot analysis is a non-quantitative method of determining what?

A

whether an Ab sample can bind to a protein antigen

28
Q

Desribe the process of running a western blot analysis.

A
  1. the antigen of interest is separated and transferred to a membrane
  2. the membrane is probed with antibody
  3. the antibody binds its specific antigen
  4. bound antibody is detected using a secondary antibody bound to enzyme
29
Q

Is ELISA a quantitative or qualitative method of measuring antigen-specific antibody?

A

quantitative

30
Q

What are the steps of an ELISA?

A
  1. put antigen on a plate
  2. add antibody; it binds antigen
  3. add secondary antibody with conjugated enzyme; it binds the Ab:Ag complex
  4. add chromogen; enzyme reacts with chromogen and ligand is visualized
    The color change can be quantitated by spectrophotometry
31
Q

What are the major benefits and limitations of ELISA?

A

benefits: very sensitive, quantitative, high throughput, cheat
limitations: requires either purified antigen or monoclonal Ab

32
Q

What are the major benefits and limitations of Western blot?

A

benefits: purified Ag or Ab not needed; highly sensitive
limitations: non-quantitative

33
Q

A serum sample from an immunized animal is commonly called ____ ____ because _____.

A

polyclonal antiserum because a complex mixture of antibodies can be elicited by one immunogen (antigen) since most immunogens have multiple epitopes

34
Q

A flow cytometer is used to rapidly analyze cells based on what 2 features?

A
  1. physical characteristics: size and granularity–measured by light scattering
  2. surface marker expression–measured by fluorescence emissions when cell is conjugated to a fluorochrome
35
Q

The following types of light scatter in flow cytometry are influence by what?:

  1. forward scatter
  2. side scatter
A
  1. forward scatter is influenced by size and refractive index
  2. the more irregular or granular a particle is, the more side scatter
36
Q

A ____ ____ is a special flow cytometer that collects cells with the desired surface phenotype after they have passed through the laser.

A

cell sorter

37
Q

Immunohistochemistry is a way to visualize tissue sections. Please describe how.

A

uses antibodies that are conjugated to particles that can be visualized; can see tissue sections stained this way on light or electron microscopy

38
Q

Immunofluorescence is a way to visualize tissue sections. Please describe how.

A

uses antibodies that are conjugated to fluorochromes; visualized with a fluorescent microscope

39
Q

What is an agglutination titer?

A

it’s the highest dilution of a serum which causes clumping of microorganisms or other particulate antigens (often RBCs)

40
Q

What is a serial dilution?

A

repeated dilution of a sample by the same dilution factor; typically performed in a microtiter plate

41
Q

What is convalescent serum? (aka convalescent-phase serum)

A

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

42
Q

What is acute phase serum?

A

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

43
Q

What does ELISA stand for?

A

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)

CMOD- Block1 (36 decks)

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