Determining the Reactivity and Titre of Serum using a Haemagglutination Assay.

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by  Costabile, Maurizio

JournalofVisualizedExperiments www.jove.com
VideoArticle
Determining the Reactivity and Titre of Serum using a Haemagglutination Assay
Maurizio Costabile
SchoolofPharmacyandMedicalSciences,UniversityofSouthAustralia
Correspondenceto:[email protected]
URL:http://www.jove.com/index/Details.stp?ID=1752
DOI:10.3791/1752
Citation:Costabile M.(2010).DeterminingtheReactivityandTitreofSerumusingaHaemagglutinationAssay.JoVE.35.http://www.jove.com/index/Details.stp?ID=1752,doi:
10.3791/1752
Abstract
Haemagglutinationisaspecificformofagglutinationandisusedwhenantibodiesbindtoredbloodcells,whichactasaparticulateantigen.Red
bloodcellsareparticularlyusefultargetsastheyarereadilyavailableandagglutinationisobservableusingthenakedeye.Thistechniqueis
commonlyusedtodeterminethetitreofanantibody(Ab),forbloodgroupingandviralquantification.Inthisvideo,thestepsinvolvedinpreparing
andperformingahaemagglutinationassayisdemonstratedusingantibodiesspecifictobloodgroupA-antigensaddedtoredbloodcells
(Revercells).Theantiserumisseriallydilutedina96wellU-bottommicrotitretray,towhichisaddedasuspensionofRevercells.Thesamplesare
mixedandthenincubatedat37°Cfor60minutes.Afterthistime,thesamplescanthenbeeasilyscoredforve,+veandintermediate(-/+)
haemagglutinationreactions.Thisapproachallowsforthereactivityandtitreofaserumsampletobeassessedusingarapidandsimpletechnique.
Thevideowillcoverthetheorybehindtheassay,howtheresultsarereadandinterpreted,howthetitreisdetermined,howtheassaycanbe
modifiedandanyissuesassociatedwiththeuseofthistechnique.
Protocol
Preparation of 5x Veronal Buffered Saline (VBS)
1. TopreparetheVeronalBufferedSaline(VBS),threeseparatesolutionsneedtobeprepared.
2. Preparesolution1bydissolving21.25gmofNaCland0.94gmofSodiumBarbitonein350mlofdistilledwater.Thefinalconcentrationsofthe
NaClandSodiumBarbitoneare1.02Mand13mMrespectively.
3. Preparesolution2bydissolving1.44gmofBarbitonein125mlofHotdistilledwater.ThefinalconcentrationofBarbitoneis62.5mM.
4. Preparesolution3bydissolving20.33gmofMgCl2and4.41gmofCaCl2in100mlofdistilledwater.ThefinalconcentrationofMgCl2andCaCl
2is2.18Mand440mMrespectively.
5. Mixsolutions1and2andcooltoroomtemperature.
6. Oncethecombinedsolutionhascooled,add1.25mlofsolution3andadjustthepHto7.3-7.5using1MHCl.
7. Adjustthefinalvolumeto500mlwithdistilledwatertopreparea5xstocksolution.
8. Topreparea1xworkingsolution,dilutethestock1:5withdistilledwater.
Dilution of Revercells
1. GentlyresuspendthestocksolutionofRevercellsbyinversionandthenremove1mlofcellsandcentrifugefor5minat600gat4°C.
2. Aftercentrifugation,carefullyremovethesupernatantandresuspendthecellsin3mlof1xVeronalBufferedSaline.
3. Thecellscannowbestoredat4°Cuntiluse.
Procedure
1. Transfer50mlof1xVBSintorowsA1-A12ofa96wellUbottomplate.
2. ToWellA1,add50μlofanti-Aserumandmixthoroughlyusingapipette.
3. Usingafreshpipettetip,remove50μlofliquidfromwellA1andtransferittowellA2.Mixthoroughlyandrepeatthisprocessuntilyoureach
wellA11andthendiscardthelast50μlfromthiswell.
4. Theserumsamplehasnowbeenseriallydiluted2-foldfrom1:2to1:2048.NoserumisaddedtoWellA12asthisistheVBSnegativecontrol.
5. Ifmorethanoneserumsampleistobeassessed,repeatsteps4.1-4.4forrowBetc.
6. Onceallthedilutionshavebeenmade,add50μlof1%Revercellstoallwells.Note:TheRevercellswillsettleovertimeandshouldbegently
suspendedpriortobeingdispensedintothewells.
7. Mixthesamplesbygentlytappingonthesideofthemicrotitretray,coverandleavethehaemagglutinationreactiontoproceedfor1hat37°C.
Anincubatororappropriatelyheatedroomcanbeused.
8. Followingincubation,carefullyremovethetrayfromtheincubatorandexaminetheplateforhaemagglutination.TheVBScontrolwellshould
bethefirstwellexamined.Ifthereactionisnegative,thentheresultsarevalid.IftheVBSsampleshowsapositivehaemagglutinationresult,
thentheresultscannotbeusedandtheprocessshouldberepeated.Apositivehaemagglutinationreactionwillappearasabroadsheeton
thebaseofthewell,whileanegativereactionwillappearasasmallconcentratedpelletofcellsinthecentreofthewell.Anintermediate
resultwillhaveahaloofpositivecellswithacentralcoreofpelletedcells.ThisresultoccursastheserumhassomeAbthatcanreact,but
insufficientamountsofAbtocauseafullreaction.
9. Therepresentativeresultssectionhastwodiagrammaticrepresentationsoftheexpectedresults.Anexampleofaninvalidandvalidsetof
resultsisprovided.Thetitrefortheanti-serumwillalsobedetermined.
Representative Results:
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Thevideoincludesanexampleofrepresentativeresults.Below(Fig1andFig2)aretwoexamplesshowninadiagrammaticmanner.Thefirst
exampleisofareactionthatcannotbeinterpretedastheVBScontrolispositive,whilethesecondisavalidassaythatallowsforthetitretobe
determined.
Figure1.Anexampleofahaemagglutinationwherethepositiveresulthasfailed.Inthediagrammaticrepresentationabove,the
heamagglutinationreactioncannotbeaccepted.TheVBSserumnegativecontroldisplaysapositivehaemagglutinationreactionasevidencedby
thesheetformationatthebaseofthewell.Thisindicatesthatanerrorhasoccurredandtheresultscannotbeaccepted.Inthiscase,theprocess
shouldberepeated.
Figure2.Anexampleofahaemagglutinationwherethepositiveresulthasworkedcorrectly.Inthediagrammaticrepresentation,the
heamagglutinationreactionisvalid.TheVBSnegativecontroldisplaysanegativehaemagglutinationreactionasevidencedbythepelletthathas
formedatthebaseofthewell.Thelastpositivewellisrow7,whichindicatesthatthetitreforthisserumsampleis128(i.ethereciprocalof
1/128).Thiswouldbeanexampleofanantibodythatispresentinmoderateamountsintheserum.
Discussion
Thehaemagglutinationassayisaverysimpletechniquethatallowsforalargenumberofserumsamplestobeassessedwithinashortperiodof
time.Italsohastheadvantageofbeingreadwithouttheneedforspecialisedequipment,withtheeyebeingsuitable.Thistechniquecanalsobe
modifiedforthedetectionofhumanchorionicgonadotrophinhormonepresentinurine,aspartofahaemagglutination-inhibitiontest.Inrelationto
astandardhaemagglutinationtest,thereareafewareaswherecareneedstobetakeninorderfortheassaytobeperformedcorrectly.Firstly,
thistechniqueisbasedonthesettlingofcellsontoaconcavesurface.Assuch,itisessentialthataU-orroundbottommicrotitreplateisused.
TheuseofaflatbottomplateasiscommonlyusedforELISAisnotsuitable,asnegativeandpositivereactionswillappearidentical.Itshould
alsobeappreciatedthatwhendifferentserumsamplesareused,reactiveantibodiesarelikelytobepresentatdifferingconcentrations.Athigh
antibodyconcentration,theantigen(Revercells)maybelimitingandaprozoneeffectmaybeobserved.Howeverthiswillnotaffectthe
determinationofthetitrevalue,sincethisisdeterminedasbeingthelastwellwhereapositivereactionwaslastseen.Lastly,thistechniqueisnot
quantitative,theabsolutelevelofreactiveantibodyintheserumisnotknown.However,inmostcasesthisisnotnecessary,particularlywhenthe
determinationofreactivitytoaknownantigenisalltheinformationthatisrequired.
Acknowledgements
TheauthorwouldliketothankMissLoraMatthewsforperformingthetechniqueandMrPaulShepherdforcinematography.Thisprojectwas
fundedbytheUniversityofSouthAustralia,LaboratoryMedicineprogram.
References
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