Medical Microbiology

peptidecantheoreticallybeusedtoassayspecificCD4+Tcells,butarestill

difficulttomanufacture.Usingthetetramertest,specificTcellscanbede-

tecteddirectlyfrombloodorlymphoidorgans.Histologicalapplications

arefeasible,butstilldifficult.

LymphocyteFunctionTests...........................

Certainfunctionsofisolatedlymphocytepopulationscanbedeterminedbya

numberofmethods:

&Determinationofthenumberofcellsproducingantibodies,e.g.,thehemo-

lyticplaqueassayinwhichantibodyproductionistestedbyaddingantigen-

couplederythrocytes.Inthevicinityofantibody-secretingcells,theerythro-

cytesarecoveredwithantibodiesandcanbelysedbyadditionofcomple-

ment.Today,ELISAmethodsaremoreoftenusedthanerythrocytes(ELISPOT).

&ELISPOTASSAY:usedtomeasureantibody-producing,orIL-releasing,lym-

phocytes.Theantigenoranti-ILantibodyisfixedonaplasticsurface.Lym-

phocytesarethenplacedoverthis,withinathinlayerofagarmedium.When

thecellsareincubatedat 378 C,theymaysecretetheantibodiesorILrecog-

nizedbythecorrespondingtestsubstances.Afteracertainperiodoftime,the

celllayerisshakenoffandthepreparationisthoroughlywashed.Thebound

materialcanthenbedevelopedusinganoverlaidsemisolidagar,asforthe

ELISAmethod.Theenzymereactiongeneratesspotsofcolor,eachofwhich

correspondstoacell,andwhichcanbecounted(Fig.2. 30 ).

&Measurementofthereleasecapacityofcytokines,ordetectionofmRNA,is

alsopossiblewiththeELISPOTassay.

&Lymphocytestimulationassay:isolatedlymphocytesareincubatedwith

antigeninculturemedium.Measuringthe^3 H-thymidineincorporation,

132 2 BasicPrinciplesofImmunology

Fig.2. 31 Thisdeviceanalyzescellsbymeansoffluorescent-labeledantibodies

directedagainstcellsurfaceantigens–orforpermeabilizedcells,directedagainst

internalcellantigens.Intheexampleshown,peripheralbloodlymphocytes(PBL)

areincubatedwithmonoclonalantibodiesspecificforCD4orCD8,resultinginthe

distributionoffluorescenceintensityasindicatedina.Inbthelabelingofdifferent

cellpopulationswithanti-CD4oranti-CD8isshown.Bythismeans,thepercen-

tagesofthesubpopulationsinthetotalpopulationcanbedetermined.Thefluor-

escence-activatedcellsortershownin(c)makesuseofthisdata.Bymeansof

vibration,thecellstreamisbrokenupintofinedropletswhich,dependingon

thefluorescenceandsortingsettingsused,arechargedjustbeforetheyarese-

paratedandideallycontainonecelleach.Certainparametersaremeasuredfor

eachcellwiththehelpofalaserbeam,where-uponthedropletsaredeflected

intotheintendedcontainersbythe+and–platefields. "

2

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Kayser, Medical Microbiology © 2005 Thieme

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