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Immunology (By: Ben + Dustin) > Seminars 3/4 (Ben) - Labeled AB Tests + FCT > Flashcards

Seminars 3/4 (Ben) - Labeled AB Tests + FCT Flashcards

1
Q

What is a hapten?

How are they used in creating Abs for lab diagnostics?

A
  • small molecules that elicit an immune response only when attached to a large carrier molecule
  • carrier often also does not elicit its own response
  • hapten/carrier complexes are injected into animals to create polyclonal antibodies for hapten, carrier and hapten/carrier complex
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2
Q

What is a hybridoma and what is it for?

A

a laboratory-made hybrid myeloma/B-cell which can produce antibodies, is immortal and has a high capacity for production

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3
Q

What are the 3 different types of epitope variants?

A
  1. Conformational - only bindable by Ab when antigen is in its native conformation
  2. Linear - can be accessible or hidden when antigen is in native conformation (may require denaturation)
  3. Neoantigenic - an epitope that is created by proteolysis (is near site of lysis + exposed after cleavage)
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4
Q

What are 4 different ways that antibodies can be labeled for use in diagnostic techniques?

A
  1. Radio-isotopes - detected via scintilligraphy
  2. Colloidal gold - detected via EM
  3. Fluorochrome - fluoresce visibly
  4. Enzymes - catalyze visible color reactions
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5
Q

What is sensitivity vs. specificity in terms of immunological testing?

A
  • Sensitivity - the % of positive samples which are recognized as positive by a certain test
  • Specificity - the % of negative (“healthy”) samples which are recognized as negative
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6
Q

Describe indirect ELISA.

A

Used to test for antibody presence…

  1. Coating - coat well bottoms with antigen + wash out excess, only firmly bound Ag remain
  2. Blocking - block well sides with anti-adhesive material to prevent non-specif. Ab binding
  3. Primary Ab - add pt sample + wash out excess Abs not bound to Ag
  4. Secondary Ab - enzyme-linked Abs bind to Fc region of primary + unbound are washed out
  5. Color rxn - add substrate to create color rxn + measure
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7
Q

Describe sandwich ELISA.

A

Used to detect antigen…

  • Similar to indirect ELISA, but well is coated with Abs against Ag in question
  • Enzyme-linked secondary Abs bind to other epitopes on Ag
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8
Q

Describe competitive ELISA.

A

Used to check for antigen presence…

  1. Incubate primary Abs with sample (any antigen in sample will bind Ab to form complex)
  2. Add Ab-Ag complex solution to Ag-coated well
  3. Any unbound Abs in complex sol’n bind well Ags + rest of solution is washed out
  4. Add 2ndary Ab, wash + add color rxn substrate
  5. More color rxn = more unbound [primary Ab] in complex solution = lower [Ag] in original sample
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9
Q

Give a few examples of things that are routinely tested for using ELISA.

A
  • Indirect ELISA: Anti-Cardiolipin IgM, Anti-Gliadin IgG/A/M, Anti-HIV Abs
  • Sandwich/Competitive: PSA (prostate specific antigen), hCG, ferritin, steroid/thyroid hormones
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10
Q

Describe ELISPOT.

A

Detects antigenic secretions of living cells…

  1. Cell culture plate bottom is coated w/ primary Abs for antigenic products of cells in plate
  2. Enzyme-linked secondary Abs are added + bind to other epitopes on Ags
  3. An insoluble rxn product is formed by enzymes on 2ndary Abs + forms a visible spot
  4. Larger spot = more secreted product
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11
Q

What two ELISA/ELISPOT methods are used to test for M. tuberculosis infection + how?

A

Both look for rapid IFN-y production by memory T-cells against TB…

  • ELISA Quantiferon Test: incubate blood + 3 diff. TB antigens in 3 diff. tubes + measure released IFN via ELISA
  • ELISPOT T-Spot Test: culture memory cells + incubate with TB antigens on plate coated with anti-IFN Ab, wash off cells + measure IFN product
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12
Q

Describe IRMA.

A

Immunoradiometric Assay

  • is basically sandwich ELISA using an isotope-labeled secondary antibody against the antigen in question
  • (tube is coated with specific primary Ab, tests for presence of Ag in sample)
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13
Q

Describe RIA.

A

Radioimmunoassay

  • Radiolabeled
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14
Q

Describe immunohistochemistry.

A

Used to detect immobilized antigens in tissue sections or cultivated cell populations…

  • Primary ABs for Ag in question are applied to tissue section
  • Enzyme-linked or fluorescently-labeled secondary antibodies are applied to primary ABs
  • Either ultraviolet or normal light microscopy is used to detect fluorescence/enzymatic rxn product
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15
Q

What are two examples of uses of immunohistochemistry (given in seminar)?

(maybe not so important)

A
  1. Enzyme-linked detection of insulin secretion by beta cells in pancreas section
  2. Fluorescent detection of anti-RNP autoantibody from blood sample on cultured hepatocytes
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16
Q

What is direct vs. indirect immunohistochemistry?

A

Direct = labeled antibody attaches directly to molecule in question

Indirect = a secondary labeled antibody is used + attaches to a primary antibody which binds the molecule in question

17
Q

What is the difference between confocal and traditional microscopy?

(Give an example of where confocal microscopy can be used.)

A
  • Traditional: whole sample is illuminated and unfocused background elements enter img
  • Confocal: specific points in sample illuminated via laser, thinner sections of sample appear in image without distortion from background, scanning multiple sections can give 3D img
  • (Auto-antibodies against glomerular structures can be applied and “optical sectioning” of glomerulus via confocal microscopy can be done.)
18
Q

Describe Western blot.

A
  1. Proteins in sample are separated by SDS-PAGE
    • SDS - linearizes + equalizes (-) charges on proteins
    • PAGE - polyacrylamide gel electrophoresis
  2. PA gel is stained with coumassie blue and blotted onto nitrocellulose to preserve pattern
  3. NC membrane treated w/ primary/secondary ABs
  4. Chemiluminescent reaction catalyzed by enzyme on secondary AB is detected by photosensitive film
19
Q

Describe a lateral flow test…

A

Used to check for antigen (most commonly hCG in home pregnancy tests)…

  • Sample fluid is added to test strip containing colored latex beads coated w/ primary ABs
  • Antigen binds ABs on beads, flows to “test band” of secondary ABs which bind another epitope on the antigen, allowing complexes to accumulate, forming visible colored line
  • “Control band” further down strip binds ABs on beads to show that they flowed properly
  • (both band lines = pos, control only = neg)
20
Q

Give several examples of lateral flow tests used diagnostically in medicine.

A
  • H. pylori - IgG
  • Fecal occult blood (checks for Hb)
  • E. coli - O antigen
  • HIV (checks for Abs in saliva/blood/urine)
21
Q

What are the advantages of flow cytometry (FCM) over microscopic examination of cells?

A
  1. Higher #s examined (105-106 cells/exam.)
  2. Faster (1-2 minutes)
  3. Objectivity
  4. Reproducibility
  5. Automated Sample Prep.
22
Q

What are 5 things that are commonly checked for using flow cytometry?

A
  1. Malignant Leukemia
  2. Minimal Residual Disease - cells left over after leuk. treatment
  3. Multidrug Resistance
  4. Efficacy of Bone Marrow Transplantation
  5. AIDS
23
Q

Describe the basic steps of flow cytometry.

A
  • Separate cells from other sample components (clotting factors, RBCs, etc.)
  • Tag cells with fluoro-antibody
  • Device pulls cells in suspension through tube single-file + illuminates them with a laser
  • Labeled cells emit certain wavelength detected by detector which counts them + composes a histogram
24
Q

How can detectors placed at different angles to a flow cytometer detect different characterstics of a cell?

A
  • Forward scatter is measured to detect relative size of cell
  • Side scatter is measured to detect granularity/internal complexity of cell
25
Q

Besides size + granularity/complexity…

what cell characterstics can flow cytometry measure?

A
  • Relative Fluorescence Intensity - indicates relative presence of the surface antigen that binds fluoro-antibody label
  • Kinetics - time dependency of other parameters measured (not sure what this means, was in PPT)
26
Q

What can be used to directly bind DNA in cell cycle studies using flow cytometry?

A

Propidium Iodide

  • important in monitoring childhood leukemia (during which more cells are in proliferative S phase)
  • DNA content is relative to phase of cell cycle –> more PI binding + detection by cytometer = more DNA in cell = proliferative cycle phases
27
Q

What is the most important prognostic marker looked for with flow cytometry in acute lymphoid leukemia (“ALL”)?

A

CALLA

common acute lymphoid leukemia antigen

28
Q

What two ways can flow cytometry be used to detect multi-drug resistance?

A
  1. Immunophenotyping of MDR pumps (eg P-glycoprotein) by binding labeled ABs to them
  2. Functional studies of pumps using fluorescent pump substrates (eg Calcein​) … more pump function = more fluoro-substrate pumped out of cell + less detection of fluorescence by FCM
29
Q

Changes to what specific cells are seen in HIV infected patients?

And what are the changes?

A

CD4+ Th cells

  • normally are 20-40% of all lymphocytes
  • drops below 14% in HIV patient indicate severe immunodeficiency

Immunology (By: Ben + Dustin) (12 decks)

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