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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: Copyright©2010JournalofVisualizedExperiments Page1of2 JournalofVisualizedExperiments www.jove.com 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 Copyright©2010JournalofVisualizedExperiments Page2of2

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