Medical Microbiology

&Thedirect’primary’antibody,orthedetected’secondary’antibody,isla-

beledwithperoxidase.Followingtheantigen-antibodyreaction,largepre-

formedperoxidase-antiperoxidasecomplexesareaddedtothetissuesection;

thesecomplexescanattachtotheperoxidase-labeledantibodies,whichare

alreadyspecificallybound,thusamplifyingthesignalconsiderably(Fig.2.26c).

&Similarly,biotinylatedantibodiescanbeused.Thevitaminbiotinisbound

withstrongaffinitybyavidin,abasicglycoprotein.Variouscolorantsoren-

zymescoupledtoavidinthusfacilitatethecolorreactions.Suchreactionscan

beamplifiedonthetissuesectionbyaddingpreformedbiotin-avidin-perox-

idasecomplexesthatbindtothosebiotin-coupledantibodieswhichhaveal-

readybeenbound.

RadioimmunologicalandEnzymeImmunologicalTests

Radioimmunoassay(RIA)andenzymeimmunoassay(EIA),alsoknownas

ELISA(enzyme-linkedimmunosorbentassay)(Fig.2. 28 ),arenowused

veryfrequentlytotestforantigensandantibodies.Allabsorbencytestsin-

volvethefixationofantigensorantibodiestoaplasticsurface.Thelowerde-

tectionlimitisafewnanograms.Thismethodformsthebasisofmodernhe-

patitisserology,HIVtests,andtestsforautoantibodies,lymphokines,cyto-

kines,etc.Alloftheseassayscanbeperformedinadirectform(different

sandwichcombinationsofantigen,antibodyandanti-antibody,Fig.2. 27 )

128 2 BasicPrinciplesofImmunology

Fig.2. 27 Forsolidphasetestsboththeantigenandantibodyareboundtoasolid

phase(e.g.,plasticsurface).Variousmethodsarethenusedtodetectanyinter-

actionbetweentheantigenandantibody.Inthedirecttest(a)animmobilized,

unknown,antigencanbedetectedusingafluorescent-labeledantibody.Iftheim-

mobilizedantigenisknown,thistestmethodcanalsobeusedtodetectananti-

bodyboundtotheantigen.Inthesandwichmethod(b)aknownantibodyisim-

mobilized.Detectionofantibody-antigenbindingisthenperformedusinga

second,labeledantibodywhichinteractswiththeantigenatadifferentsite.

Thecapturemethod(c)canbeusedtodetectanyantigen,forinstanceIgManti-

bodies.First,anti-IgMantibodiesareimmobilized,thenserumcontainingIgMis

addedtothem.TheboundIgMcanthenbindaforeignantigen(e.g.,avirus).The

detectionprocedurenextmakesuseofeitherthelabeledforeignantigenoraspe-

cific,additionallylabeled,antibodywhichbindstotheboundantigenbutnotto

theplasticboundantibody.Inthecompetitionorcompetitiveinhibitiontest(d)

antibodiesareimmobilized,andlabeledantigensarethenboundtothem.Anun-

labeled(unknown)antigenisadded,whichcompeteswiththelabeledantigen.

Thelevelofinteractionbetweentheantibodyandtheunknownantigenis

thendeterminedbymeasuringattenuationofthesignal. "

2

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Kayser, Medical Microbiology © 2005 Thieme

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