Medical Microbiology

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munefactorspresentwithinpatientcellsandtissues.Forthispurposetissue
sections,orcellpreparations,aretreatedwithspecificantibodies(anti-sera)
whichhavebeenlabeledwithafluorochrome(Fig.2.26a).Antigen-antibody
reactionscanthusbedetectedusingafluorescencemicroscope.Thefluoro-
chromeabsorbslightofacertainwavelength(e.g.,UVlight),andemitsthe
lightenergyintheformoflightatadifferent(visible)wavelength.The
fluorochromefluoresceinisothiocyanate(FITC),whichabsorbsUVlight
andemitsitasgreenlight,isusedmostfrequently(caution:bleachesout
quickly!).

126 2 BasicPrinciplesofImmunology

Hemagglutination
Erythrocyte
antigen

Antigen
artificially fixed
on erythrocyte

Reciprocal serum dilution

Test serum a
positive
Test serum b
negative
Test serum c
positive 1/8
with prozone 1/2

2 4 8 16 32 64 128 256 5121024

Control
pos. neg.

(^1 32)
Fig.2. 25 Thehemagglutinationtestisbasedontheprinciplethaterythrocytes
cross-linkedbyantibodiessettletothebottomofthemicrotiterplatewellsinmat-
likeaggregates(testseraaandc),whereasnon-agglutinatederythrocytescollect
atthelowestpointofthewellstoformasingle“button”inthemiddle(test
serumb).Thetestseraarefirstpipettedintothewellsattheindicateddilutions,
thentheerythrocytesuspensionisadded.Non-specificagglutinationisprevented
byadditionofanirrelevantprotein.Thetestcanbecarriedoutusingerythrocyte
antigens(aboveleft).Alternatively,otherantigenscanbefixedtotheerythrocyte
surfaceandtheagglutinationmonitored(aboveright).Theso-called“prozone”
phenomenonresultsfromnon-specificblockingmechanismspresentinsera
whichhasnotbeensufficientlydiluted.
2
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Kayser, Medical Microbiology © 2005 Thieme
All rights reserved. Usage subject to terms and conditions of license.

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