Medical Microbiology

Indirectimmunofluorescenceandenzymehistology.Inthistechniquethe
specificor“first”antibodycanbeunlabeled.Theantigen–antibodycom-
plexesthatformarethendetectedusingalabeledor“second”antibody,di-
rectedagainstthefirstantibody(Fig.2.26b).Insteadoffluorochromes,enzy-
me-labeledantibodiesarenowfrequentlyusedfortissuesections.Theen-
zymecatalyzestheformationofacolorsignalfollowingadditionofapre-
viouslycolorlessdetectorsubstance.Thiscolorprecipitateallowsthedirect
observationofsignalsusingalightmicroscopic,andexhibitslittlebleaching.
Indirectimmunofluorescencecanbeusedforthequalitativeandquanti-
tativeanalysisofantibodiesdirectedagainstparticularmicrobialantigens,or
self-tissueantigens,withinapatientsserum.Inthequantitativetest,theanti-
genisfixedinawellortoatissuesectiononaslide.Thepatientsampleis
repeatedlydilutedbyafactoroftwoandaddedtotheantigenorsectionthen
renderedvisiblewithalabeledanti–antibody.
Therearetwomainmethodsofamplifyingtheimmunohistologicalcolor
signal:

ImmunologicalTestMethods 127

AntigenDetectionMethods

Fig.2. 26 Immunofluorescence(a,b)isparticularlysuitableforthedetectionof

antigens,orspecificantibodies,fixedonplastic(solidphase)(ELISA)orpresent

withinatissuesection(immunohistology).Fordirectimmunofluorescence(a)

thespecificprimaryantibodyislabeledwithafluorochrome,oranenzyme(ELISA

=enzyme-linkedimmunosorbentassay).Thetermindirectimmunofluorescenceis

usedwhenitisnottheprimaryantibodybeingdetected,butasecondaryantibody

whichisdirectedagainsttheunlabeledprimaryantibodyandhasalsobeenlabeled

withafluorochromeorenzyme(b).Inmostcases,thismethodachievesacertain

degreeofamplification.However,anevenhigherlevelofamplificationcanbe

achievedusingpreformedcomplexesofsecondaryantibodyandenzyme(c).

Fortheperoxidasemethodthedetectorenzymeisbounddirectlytothesecondary

antibody(peroxidasecatalyzesacolorreaction).Inthebiotin-avidinmethodthe

detectorenzymesarecoupledtoeitherbiotinoravidin.

2

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Kayser, Medical Microbiology © 2005 Thieme

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