Medical Microbiology

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.