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9 Cards in this Set
- Front
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Abs utilized as probes for a variety of epitopes; Abs or Ags they detect can be labeled with:
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radioisotopes
fluorescent molecules enzymes heavy metals many others these Abs can be used in various assays to detect Ags |
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Radioimmunoassay
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radionuclide like i125, to label primary or secondary Ab or Ag
direct: primary Ab is radiolabeled & incubated with Ag; unbound Ab is washed away and bound radioactivity determined indirect: primary Ab that has bound to g is detected with radioabeled, anti-Ig (secondary Ab) - Ab-Ag complex is washed free of unbound Abs; bound radioactivity determined |
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ELISA
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assay generally performed in protein
soluble Ag is added and non-covalently binds to plastic unbound Ag washed from well unlabeled primary Abs added to well & allowed to bind unbound primary Abs washed from well enzyme labeled anti-Ig Ab's added and allowed to bind unbound enzyme labeled Abs are wshed from well an enzyme cleavable, chromogenic subsrate added to well & allowed to incubte color change indicates presence of enzyme labeled secondary Ab. b/c 2nd Ab only binds to primary Ab & primary Ab only binds to epitope, degree of color change indicates amount of epitope detected |
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variations of ELISAs
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competitive assay: label Ag coat Ab to plate; add label ; inhibit with test Ag
2 site capture assay: test strip withAb to strep; if binds wash and put in anti-strep tube & see if change color = strep |
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Fluorescent immunosorbent assay
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assay may be performed in protein abosrbing, 96 well polystyrene plates
soluble Ag added & noncovalently binds to plastic. unbound Ag is washed from well unlabled primary Abs added to well & allowed to bind unbound primary Abbs washed fluorochrome labeled anti-labeled anti Ig Abs are added to well & allowed to bind unbound labeled Abs washed from well fluorescence indicates presence of epitopes bead arrays that us this technology: can do 25 diff Ags at same time |
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immunofluorescence
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fluorochrome labeled Abs used to visualize eptiopes on cells & tissues by microscopy. tissue sections or cells affixed to glass microscope slides
indirect & direct |
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flow cytometry
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single cell suspensions of lukocytes are prepared and stained with fluorescent dye labeled Abs
labeled cells contained within sheath fluid pass single file through vibrating nozzle, where laser beam passes through stream b/4 droplets are formed. cells refract & reflect light photodetector measures refracted ligh or forward scatter & measure of cell volume reflected light or side scatter, measured at right angles to laser beam, is an indication of cellular granularity. beam splitters pass reflected light through filters to photomultilier tubes to measure green or red fluorescence signals generated are analyzed by computer & represented graphically on screen foward & side scatter data allow flow cytomer operator to distinguish cells on basis of their morphology & electornicaly gate pops for further analysis ID'd populations can then be isolated or sorted : anode/cathode |
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immunoblotting
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Ag samples first separated in an analytical gel.
resolved molecules are transferred electrophoretically to a nitrocellulose membrane in blotting tank blot treated with Ab to specific Ag, washed, and radiolabeled conjugate to detec Abs is bound to blot after washing again, blot placed in contact with x ray film Ag bands which have bound Ab are visible based on size; big molecules stay on top and small molecules migrate to bottom |
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proliferation assays
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to determine ability of lymphocytes to respond to a stimulus
can be coupled with Quantiferon-TB gold & quantiferon-TB gold in tube Ag (for TB) or mitogen are added to drawn whole blood, allowed to incubate 16-24 hours, tested by ELISA for IFN-g, TB is Th1 initiator - causes t cells to proliferate, become Th1 cells & secrete IFN-g - if no IFN-g detected, then pt has not seen TB highly sensitive & specific may replace PPD & x-ray |
