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IMMUNOLOGY: CMOD-Week 3 > (11) Immunoassays > Flashcards

(11) Immunoassays Flashcards

1
Q

Do the IgG subtypes differ in their avidity potential for cognate antigen?

A

*To me it doesn’t appear that they would because they all appear monomeric, but Dr. Miller implied that that they might

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2
Q

What is an immunodiffusion assay and when would you use it?

A

QUALITATIVE test with POOR sensitivity (20µg - 2mg/mL) to look whether or not antibodies are binding a particular antigen to look for precipitates that form

**Useful for SIMPLE mixtures only

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3
Q

what is Immunoelectrophoresis?

A

QUALITATIVE test with POOR sensitivity (20µg - 2mg/mL). Uses electophoresis to separated out serum and see whether or not certain things are present (e.g. looking for certain Ig’s in the blood of a normal person compared to BTK deficient person)

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4
Q

Hemagglutination

  • Basic Principal
  • Qualitative or Quantitiative
A
  1. RBCs are used to bind to immune complexes in a 96 well serial dilution
  2. RBC’s that bind indicate a positive test and form a red blob on the floor of the plate
  3. RBCs that don’t bind are negative and form a dot

**easily visualized

**Better Sensitivity (less than 1µg/ml)

**QUANTITATIVE

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5
Q

What is a direct Coomb’s test (DAT)?

  • what is it used for?
  • Qualitative or Quantitative?
A

Test that detects antibodies or complement proteins on the surface of RBCs collected directly from a patient

(e. g. test given to pt. w/ hemolytic anemia to see if its autoimmune)
1. Patients blood is taken and washed to remove serum with unbound complement or antibody
2. add Coomb’s reagent that contains antibodies that are specific for human Igs and complement proteins
3. If RBCs have antibodies or complement on their surface they will agglutinate and RBCs will crosslink

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6
Q

What is an indirect Coomb’s test?

  • when is it used?
  • Qualitative or Quantitative?
A

WHEN:
1. Used for cross-matching prior to transfusions
2. Used to detect atypical antibodies in mother’s serum
PREVENTION OF HEMOLYTIC DISEASE IN NEWBORN

HOW:
1. SERUM from patient needing transfusion is mixed with RED BLOOD CELLS of donor.

  1. Any exisiting blood group-specific antibodies from the recipient patient’s serium will bind to the RBC’s from the donor
  2. If the PATIENT’s SERUM has antibodies for the DONOR RBC’s then agglutination will occur

QUANTITATIVE TEST

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7
Q

What is an Monospot test?

A
  • Test is run in the same way as an indirect Coomb test except horse RBCs are used
  • Tests for EPSTEIN-BARR VIRUS
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8
Q

Complement Fixation Assay?

- Sensitivity?

A
  1. Add antibody to antigen in a mixture
  2. Add complement proteins to the mixture
  3. Add RBC’s to mixture
    - if the antibody bound the antigen, then complement bound to the antibody, not complement cascade will be triggered
    * *RBC in tact for a positive test
  • if antibody did not bind then complemet roams free and will form MAC on the added RBC
  • *RBC will lyse for negative test
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9
Q

Primary Antibody

A
  • Test sample that may or may not contain antibody specific for a particular antigen
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10
Q

Secondary Antibody

A
  • An antibody that will detect the primary antibody; typically conjugated to an indicator molecule
  • Often this is an enzyme or a fluorochrome
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11
Q
  • *Convalescent Serum (or convalescet-phase serum)

- Relative amounts of antibody types present in this serum

A

Serum from a person who has recuperated from a particular infection (e.g. scarlet fever, which may be of use in treating a person with the same infection

**LOW IgM, and HIGH IgG

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12
Q
  • *Acute-phase serum:
  • Relative amounts of antibody types present in this serum
  • other protein types present
A

Serum from a patient that is actively infected with a pathogen

  • *HIGH IgM, and LOW IgG
  • *Often contains high levels of MANNOSE BINDING PROTEIN and C-Reactive protein and FIBRINOGEN
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13
Q

What happens on the cellular level during immediate (type 1) hypersesitivity reactions?

A
  • IgE that is bound to mast cell (via Fc(epsilon)RI) cross-links and causes mast cell degranulation
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14
Q

What is RAST and what is it used for?

A
  • ELISA-type assay except the antibodies have been radiolabled amplifies the signal given from the small amount of IgE that can be found in the blood

***This test has to be extremely sensitive because there is so little IgE in the blood

***This method got replaced around 2010

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15
Q

What is ImmunoCAP and what is it used for?

A
  • ELISA-type assay that uses fluorescence instead of radiolabel, this eliminates the pain of dealing with radioactive materials.
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16
Q

What are the steps to performing a Western Blot?

A
  1. Do electrophoresis to separate protein onto polyacrimide
  2. Transfer proteins to Nitrocellulose
  3. Add antibody of interest and wash
  4. Add anti-antibody antibody that is labeled
  5. Shoot films to see where the anti-antibody antibody is bound to determine which proteins are actually bound by the antibody of interest
17
Q

What does immunohistochemistry look for?

A

Specific antigens within a tissue or even a single cell

18
Q

How is immunohistochemistry performed?

A
  • Some kind of visualization particle is attached to a an Ab (gold or flurochrome), we can this visualize where things are binding and what is happening at particular times in the cell
    (e. g. Mast Cells bound with Ag labeled IgE, CTLs with fluorescent microtubules)
19
Q

What is FACS?

- when would you use it?

A

Fluorescence-Activated Cell Sorting

*Flow cytometry paired with a sorter

**Could use FACS to sort out CD34+ stem cells and use them in a bone marrow transplant

20
Q

What is flow cytometry?

A
  1. Label cells with antibodies that have fluorescent tags of different colors
  2. Shoot a lazer at the cells one at a time and measure:
    - forward scatter
    - side scatter
    - Color (with photomultiplier tube)
  3. Forward scatter (proportional to size) can be plotted against side scatter (proportional to surface irregularity) to determine if correct cells have been isolated
  4. Color is measured and it tells you how many of cells have been labeled with a particular antibody (this can be indicative of cell type etc.)

IMMUNOLOGY: CMOD-Week 3 (3 decks)

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