Medical Microbiology

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marrowtransplants.ThistestisbasedontheprinciplethatTlymphocytesare
stimulatedtoproliferatebynonselfMHCclassIorIIantigensandtodevelop
intocytotoxicTcellsdirectedagainstclassI.
&Chromiumreleaseassaymeasurescytolyticactivity,mainlybyCD8+Tcells,
directedagainstallogeneic,virus-infected,orpeptide-loadedtargetcells.The
targetcellsareincubatedwith^51 Crwhichthecellsincorporate.Theyare
thencultivatedwitheffectorcellsfor4–6hours.Whenthetargetcellsare
lysedchromiumisreleasedintotheculturemedium,followingwhichit
canbequantitativelymeasured.
&Assayofintracellularcytokines.Followingabriefstimulatoryculture
(sixhours),thecellsarerenderedpermeableusingamilddetergentso
thatspecificallylabeledantibodiescandiffuseintothecells.Labeledcells
canthenbeanalyzedbyFACSequipment(orbyamicroscope).

134 2 BasicPrinciplesofImmunology

TetramerAssay

T cell

T cell

T cell

Avidin with 4
binding sites for
biotin

Biotin

Specific peptide

Labeling of avidin
with fluorescein,
phycoerythrin, etc.

MHC class I
β2-microglobulin

Fig.2. 32 Thetetramerassayisanantigen-specificbindingtestforlivingTcells.
Complexescomprisingbiotin-coupledMHCclassIheavychains,b 2 microglobulin,
andspecificpeptideareproperlyfolded,washed,thenboundtoavidin(whichcon-
tainsfourbindingsitesforbiotin).Theresultingtetramericcomplexesarethen
incubatedwithapopulationofTcells.ThoseTcellsexpressingtheappropriate
T-cellreceptorwillbindtotwoorthreeoftheexposedMHCclassI-peptidecom-
plexespresentoneachtetramer.Labelingoftheavidincomponentwithfluores-
cein,phycoerythrin,orotherfluorescentsubstancesthenpermitsFACSanalysisof
tertramerbindingTcells.

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Kayser, Medical Microbiology © 2005 Thieme
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