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
