Seminars 3/4 (Ben) - Labeled AB Tests + FCT Flashcards
What is a hapten?
How are they used in creating Abs for lab diagnostics?
- 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
What is a hybridoma and what is it for?
a laboratory-made hybrid myeloma/B-cell which can produce antibodies, is immortal and has a high capacity for production
What are the 3 different types of epitope variants?
- Conformational - only bindable by Ab when antigen is in its native conformation
- Linear - can be accessible or hidden when antigen is in native conformation (may require denaturation)
- Neoantigenic - an epitope that is created by proteolysis (is near site of lysis + exposed after cleavage)
What are 4 different ways that antibodies can be labeled for use in diagnostic techniques?
- Radio-isotopes - detected via scintilligraphy
- Colloidal gold - detected via EM
- Fluorochrome - fluoresce visibly
- Enzymes - catalyze visible color reactions
What is sensitivity vs. specificity in terms of immunological testing?
- 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
Describe indirect ELISA.
Used to test for antibody presence…
- Coating - coat well bottoms with antigen + wash out excess, only firmly bound Ag remain
- Blocking - block well sides with anti-adhesive material to prevent non-specif. Ab binding
- Primary Ab - add pt sample + wash out excess Abs not bound to Ag
- Secondary Ab - enzyme-linked Abs bind to Fc region of primary + unbound are washed out
- Color rxn - add substrate to create color rxn + measure
Describe sandwich ELISA.
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
Describe competitive ELISA.
Used to check for antigen presence…
- Incubate primary Abs with sample (any antigen in sample will bind Ab to form complex)
- Add Ab-Ag complex solution to Ag-coated well
- Any unbound Abs in complex sol’n bind well Ags + rest of solution is washed out
- Add 2ndary Ab, wash + add color rxn substrate
- More color rxn = more unbound [primary Ab] in complex solution = lower [Ag] in original sample
Give a few examples of things that are routinely tested for using ELISA.
- Indirect ELISA: Anti-Cardiolipin IgM, Anti-Gliadin IgG/A/M, Anti-HIV Abs
- Sandwich/Competitive: PSA (prostate specific antigen), hCG, ferritin, steroid/thyroid hormones
Describe ELISPOT.
Detects antigenic secretions of living cells…
- Cell culture plate bottom is coated w/ primary Abs for antigenic products of cells in plate
- Enzyme-linked secondary Abs are added + bind to other epitopes on Ags
- An insoluble rxn product is formed by enzymes on 2ndary Abs + forms a visible spot
- Larger spot = more secreted product
What two ELISA/ELISPOT methods are used to test for M. tuberculosis infection + how?
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
Describe IRMA.
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)
Describe RIA.
Radioimmunoassay
- Radiolabeled
Describe immunohistochemistry.
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
What are two examples of uses of immunohistochemistry (given in seminar)?
(maybe not so important)
- Enzyme-linked detection of insulin secretion by beta cells in pancreas section
- Fluorescent detection of anti-RNP autoantibody from blood sample on cultured hepatocytes
What is direct vs. indirect immunohistochemistry?
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
What is the difference between confocal and traditional microscopy?
(Give an example of where confocal microscopy can be used.)
- 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.)
Describe Western blot.
- Proteins in sample are separated by SDS-PAGE
- SDS - linearizes + equalizes (-) charges on proteins
- PAGE - polyacrylamide gel electrophoresis
- PA gel is stained with coumassie blue and blotted onto nitrocellulose to preserve pattern
- NC membrane treated w/ primary/secondary ABs
- Chemiluminescent reaction catalyzed by enzyme on secondary AB is detected by photosensitive film
Describe a lateral flow test…
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)
Give several examples of lateral flow tests used diagnostically in medicine.
- H. pylori - IgG
- Fecal occult blood (checks for Hb)
- E. coli - O antigen
- HIV (checks for Abs in saliva/blood/urine)
What are the advantages of flow cytometry (FCM) over microscopic examination of cells?
- Higher #s examined (105-106 cells/exam.)
- Faster (1-2 minutes)
- Objectivity
- Reproducibility
- Automated Sample Prep.
What are 5 things that are commonly checked for using flow cytometry?
- Malignant Leukemia
- Minimal Residual Disease - cells left over after leuk. treatment
- Multidrug Resistance
- Efficacy of Bone Marrow Transplantation
- AIDS
Describe the basic steps of flow cytometry.
- 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
How can detectors placed at different angles to a flow cytometer detect different characterstics of a cell?
- Forward scatter is measured to detect relative size of cell
- Side scatter is measured to detect granularity/internal complexity of cell
Besides size + granularity/complexity…
what cell characterstics can flow cytometry measure?
- 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)
What can be used to directly bind DNA in cell cycle studies using flow cytometry?
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
What is the most important prognostic marker looked for with flow cytometry in acute lymphoid leukemia (“ALL”)?
CALLA
common acute lymphoid leukemia antigen
What two ways can flow cytometry be used to detect multi-drug resistance?
- Immunophenotyping of MDR pumps (eg P-glycoprotein) by binding labeled ABs to them
- 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
Changes to what specific cells are seen in HIV infected patients?
And what are the changes?
CD4+ Th cells
- normally are 20-40% of all lymphocytes
- drops below 14% in HIV patient indicate severe immunodeficiency
