Medical Microbiology

populationscanbecoatedwithmagneticbeads,orsheeperythrocytes

loadedwithspecificantibodies,thenpurifiedusingamagnetoraFicoll

gradient.Thefluorescence-activatedcellsorter(FACS,Fig.2. 31 ,p. 133 )is

nowregularlyusedforthispurpose.Inthisassay,monoclonalantibodies

labeledwithvariousfluorochromesdirectedagainstcellsurfaceantigens

(suchasCD4,CD8),oragainstintracellularcytokines(whichinvolvesthe

useofdetergentstoincreasethepermeabilityofthecellmembrane),arein-

cubatedwiththeisolatedbloodlymphocytes.Alternatively,antigen-specific

TlymphocytescanbelabeledwithMHCclassIorIIpluspeptidetetramers

(seebelow).Followingincubation,andseveralwashingsteps,theequipment

identifiesandcountstheantibody-loadedlymphocytes,employingmagnetic

pulsesortingasrequired.

130 2 BasicPrinciplesofImmunology

RadioimmunosorbentTest(RIST)

Anti-IgE

cpm

Amount of labeled IgE*

cpm

Radioactivity

in test tube

IgE in patient serum

Amount of unlabeled IgE in IgE* test solution

Labeled IgE*

IgE from patient serum

Plastic surface

Plateau

(max. binding capacity)

Standard curve

80%

a

b

Fig.2. 28 RISTisacompetitiveradioimmunoassay(RIA)usedforquantitative

measurementofantibodiesofanygivenIgclass(inthisexampletotalIgE)within

patientserum.Anti-IgEantibodiesareallowedtoadsorbtothesolidphase(plastic

surface).Inthefirstinstance,definedconcentrationsofradiolabeledIgE(IgE*)are

usedtodeterminethemaximumbindingcapacityoftheseantibodies(a).The

actualtest(b)isthenperformedusingtheIgE*concentrationdeterminedtoresult

in 80 %saturationofthefixedantibodies:TheIgE*testsolutionisaddedtothe

fixedanti-IgEantibodiesandthepatientserumisthenaddedbypipette.Themore

IgEtheserumcontains,themoreIgE*willbedisplacedbythepatientsantibodies,

andthelowertheradioactivitylevelwillbeinthetesttube.TheIgEconcentration

inthepatientserumisthencalculatedbasedonastandardcurveestablishedpre-

viouslybyprogressively“diluting”theIgE*testsolutionwithunlabeledIgE.

2

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Kayser, Medical Microbiology © 2005 Thieme

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