Medical Microbiology

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.

2

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Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.