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IndexPage

A.1.Aim2

A.2.Principle2

B.Samples2

C.Controls2

D.1.Equipment,instrumentsand materials2

D.1.1Equipmentandinstruments2

D.1.2Materials3

D.2.Procedure4

D.2.1Cultureand synchronisation of parasites4

D.2.2UninfectedRedBloodCellpreparation5

D.2.3Preparationofthetestsamples6

D.2.4Preparationof GIAparasitesuspension7

D.2.5GIA set-up7

D.2.6GIA harvest7

D.2.7pLDHassay7

D.3.Platelay-out8

D.4.Remarks totheprocedure9

E.Results9

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A.1.Aim

ToquantifyIgG’sobtainedfrompatientsoranimalsintheirabilitytoinhibitinvasion orintraerythrocyticdevelopmentof P.falciparumintoerythrocytes.Theread-outsystem is detectionofpLDH(plasmodiumLactateDeHydrogenase).

A.2.Principle

3-AcetylpyridineAdenineDinucleotide(APAD)andLactate(presentinsubstratebuffer)areconvertedbypLDHtoAPADHandPyruvate.APADHreducesthechromogenicsubstrateNitroBlueTetrazolium(NBT)usingtheenzymediaphorase.ThisresultsintheformationofNitroBlueFormazan(NBF),adeeppurplesolublestainthatcanbemeasuredatawavelengthof650nm.

Schematicrepresentation:

APAD +

Lactate pLDHAPADH+Pyruvate

NBTDiaphorase

APAD+NBF

B.Samples

IgG’sobtainedfromhumantrialsoranimalexperiments,inwhichtheindividuals/animalswere

immunisedagainstP.falciparum.Orserafrompeoplenaturallyexposedtomalaria.

C.Controls

Positivecontrol:BG98Rbstandardat6mg/mL

Negativecontrol:Negativerabbit,rhesusorhumanIgGfraction,purifiedsimilarlyasthesamples.

D.1.Equipment,instrumentsandmaterials

D.1.1.Equipement andinstruments

1.50mlsyringes:BDPlastipak#300866

2.50mltubes:GreinerBio-one#22761

3.96-wellflat-bottomhalfareacultureplateswithindividuallids:GreinerBio-one

#675180

4.96-wellflat-bottomcultureplateswithindividuallids:GreinerBio-one#655180

5.AmiconUltraFiltertubes:AmiconUltra-15#UFC903024

6.Centrifuges:forculturingandspinningdownRBC’s:BeckmanCoulterAllegraX-22RCentrifuge

forharvestingofGIA-plates:BeckmanSpinchronRCentrifugeforIgGpurification:BeckmanCoulterAvantiJ-ECentrifuge

7.Columns:EconopackcolumnsBio-Rad#7371511;

Econo-columnFlow AdaptorBio-Rad#738-0016

8.Cultureflasks:T25:Corning#430168

T75:Corning#430720

T175:Corning#731079

9.Eppendorfcentrifuge:Eppendorfcentrifuge5424

10.Eppendorftubes:EppendorfSafe-lock1.5ml#0030120.086

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11.Filters:0.45µm:WhatmanFP30/0,45CA-S#10462100

0.2µm:WhatmanFP30/0,2CA-S#10462200

12.Flatbedshaker:EdmundBühlerTiMixCONTROL

13.Humidifiedbox:plasticboxwithwettissuesorwateratthebottom

14.Humidifiedincubator:Sanyo O2/CO2incubator

15.Microscope:ZeissAxioskop

16.Nanodrop:NanodropND-1000Spectrophotometer

17.Pipettes:2ml:ALP#PN2E1

5ml:ALP#PN5E25

10ml:ALP#PN10E25

25ml:ALP#PN25E1

18.Platereader:Bio-RadModel680MicroplateReader

19.Pump:WatsonMarlow205U

20.Screw-captubes:Sarsteadt2mlPP#72.694.006

21.Shakingincubator:innova44

22.Slides:Menzel-Gläzer76x24mm

23.Stative

24.Tips:20µl:CLP#BT20

200µl:CLP#BT200

1000µl:CLP#BT1000

25.Waterbath:Jubalo

D.1.2.Materials

1.0.3Malanine,10mMHEPESpH7.5

L-alanine:Sigma#A7469;C3H7NO2,M=89.09HEPES:Gibco#11344-025;M=238.39

Toprepare250ml0.3Malanine10mMHEPES:dissolve6.68galanineand595.75

mgHEPESin200mldistilledwater,adjusttopH7.5,fillupto250mlwithdistilledwater.Filterthroughafiltertopusingvaccuum.Storeat4°C.

2.100% Methanol:Merck#1.06009.2500;CH3OH,M=32.04

3.2xCmed=2xCompleteCultureMedium:

RPMI1640+20%Humanserum+30µg/mlGentamycin4.APAD:Sigma#A5251;C22H28N6O14P2,M=662.44

3-AcetylpyridineAdenineDinucleotide

Toprepare10mlstocksolutionof10mg/ml:dissolve100mgofAPADin10mldistilledwater.Make50µlaliquotsandstoreat-20°C.

5.Bindingbuffer:ProteinGIgGBindingBuffer:Pierce#21011

6.Cmed=CompleteCultureMedium:

RPMI1640+10%Humanserum+15µg/mlGentamycin

7.DiaphorasefromClostridiumkluyveri:Sigma#D5540

Toprepare30mlstocksolutionof50units/ml:dissolve1.500unitsDiaphorasein30mldistilledwater.Make200µlaliquotsandstoreat-20°C.

8.EDTA: Merck #1.00944.1000;C10H16N2O8,M=292.25

Ethylenediaminetetraaceticacid

Topreparean8mMEDTAsolution:Dissolve116.9mgin40mlRPMI1640,adjusttopH7.5,fillupto50mlwithRPMI1640andsterilisethrougha0.45µmfilter,storeat4°C.

9.Elutionbuffer:Pierce #21009

10.Gentamycin:Gibco#15750-037;stock=50mg/ml

11.Giemsabuffer:Merck#1.09468.0100;Na2HPO4*2H2OKH2PO4

12.Giemsastain:Sigma#GS500

TopreparefreshGiemsastain:dilute1partGiemsastainin4partsGiemsabuffer,filterthrougha0.45µmfilter,useimmediately.

13.Heat-inactivatedHumanSerumA+:BloedbankLeidsenhage,storeat-20°C.

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14.HumanRBCO+,12heparintubesofindividualdonors,afterprocessingstoreat4°C.

15.S.A.G.Msolution

Saline:SodiumChlorideSigma#S3014Adenine:Sigma#A2786

Glucose:Sigma#16325

D-Mannitol:Sigma#M9546

Toprepare500mlsolution,dissolve4.4grSodiumChloride,4.5grGlucose,0.08grAdenine,2.62grMannitolin500mldistilledwaterandsterilisethrougha0.2µmfilter.

15.plasmodiumfalciparum,e.g.FCR3or3D7strain

16.LDH-buffer:Sigma#L7022;C3H5NaO3,M=112.06

SodiumL-lactate

Toprepare500mlofthebuffer,mix50mlof1MTRIS-HCl(pH8.0)and450mldistilledwater.Add2.8gSodiumL-Lactateand1.25mlTritonX-100.Mixonamagneticstirreratroomtemparatureforatleast30minutes.Make50mlaliquotsandstoreat-20°C.

17.NaAz:Sigma#S2002;NaN3, M=65.01Sodiumazide

18.NBT:Sigma#N6639;C40H30N10O6*2Cl,M=817.64NitroBlueTetrazoliumchloride

19.PBS:Gibco#10010PhosphateBufferedSalinepH7.4

20.ProteinG:Pierce#20399

21.RPMI1640:Gibco#52400

RPMI1640+L-glutamine+25mMHEPES

22.TRIS:Merck #1.08382.2500;H2NC(CH2OH)3,M=121.14

Tris(hydroxymethyl)aminomethane

Toprepare1literof1MTRISpH9.0:Dissolve121.14gofTRISin900mlofdistilledwater,adjustpHto9.0,fillupto1literwithdistilledwater.

D.2.Procedure

D.2.1.Cultureandsynchronisationofparasites

1.*Routineculturing:

Theculturecanroutinelybemaintainedat0.1–1%parasitaemia.Thehematocritwillbekeptat5%.

Theculturemustberefreshed3timesaweek.

1.1.PlaceCmedatroomtemparaturebeforeculturing.

1.2.Takecultureoutoftheshakingincubator.

1.3.Transfer±200µlofculture-suspensiontoaneppendorf-tube.

1.4.Spinfor10secondsatmaximumspeed(~20000g)inanEppendorfcentrifuge.

1.5.Removesupernatantcarefully,leaveaboutthesameamountofsupernatantaspelletvolume.

1.6.Resuspendpelletinthesupernatantleft.

1.7.Placeasmalldropontoanslideanduseanotherslideunder±45°-angletomakeathinsmear.

1.8.Air-drytheslide.

1.9.Fixtheslidein100%methanolandairdry.

1.10.*PrepareafreshGiemsa-stain.

1.11.Filterthrougha0.45µmfilterandallow0.5mltodrainawaybeforeusingtherestofthestainforthestaining.

1.12.Stainfor5minutesatroomtemparature.

1.13.Pouroffstainsolutionandimmediatelyrinsetheslideinwater.

1.14.*Drytheslidethoroughlybeforeobservationthroughamicroscope.

1.15.Putadropofoilontheslideandestimatetheparasitaemiaunderthex100objective.

1.16.Count atleast2000RBC,incasetheyareevenlydistributedthroughouttheslide,onequarterofonefieldcanberepresentativefortheRBC-count.TogetthenumberofRBC,multiplythequarter-countofRBCby4andthanbythenumberoffieldscounted.

1.17.CountthenumberofinfectedRBCforeachfield.

1.18.%parasitaemia=(totalcountedparasites/totalRBCcounted)x100

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1.19.*Todeterminethedevelopmentalstageoftheparasitesperformadifferentialcount(rings,trophozoitesandschizontsare countedindividually).

1.20.Transferalloftheculturetoa15or50mltube.

1.21.Spinfor5minutesat900g.

1.22.Removesupernatantcarefully

1.23.TakeouttheamountofpelletneededandplacebackintothecultureflaskcontainingnewCmed.

1.24.AddRBC50%HTtoafinalhematocritof5%.

1.25.Gastheculturewith5%CO2,5%O2,90%N2,usingsterilefilters,tipsandpipettes.

1.26.Placethecultureintheshakingincubatorat37°C.

2.Synchronizationofparasiteswithalanine:

Alanine-treatmentremovesschizonts/trophozoitesfromtheculture,leavingbehindtheringsandearlytrophozoites(timewindowof0-16hours).

Beforeyoustartthealanine-treatmentyoushoulddoadifferentialcountofyourculturetomakesurethereareringsinyourculture.

Itisadvisedtorepeatthealanine-treatment2to3timesbeforeusingthecultureforaGIA.

2.1.Pre-warmasolutionof0.3Malanine,10mMHepespH7.5at37°C.

2.2.Transfertheculturetoa50mltube.

2.3.Spinfor5minutesat900g.

2.4.Removesupernatantcarefully.

2.5.Resuspendthepelletin5xpelletvolumeof0.3Malanine,10mMHepespH7.5.

2.6.Incubatefor20to30minutesat37°C.

2.7.Fillupthe50mltubewithCmed.

2.8.Spinfor5minutesat900g.

2.9.Removesupernatantcarefully.

2.10.Resuspendpelletin50mlCmed.

2.11.Spinfor5minutesat900g.

2.12.Removesupernatantcarefully.

2.13.*TakeouttheamountofpelletneededandtransfertoanewcultureflaskcontainingnewCmed.

2.14.AddRBC50%HTsothatthehematocritwillbe5%.

2.15.Gastheculturewith5%CO2,5%O2,90%N2,usingsterilefilters,tipsandpipettes.

2.16.Placethecultureintheshakingincubator.

D.2.2.UninfectedRedBloodCellpreparation

1.IsolationofRBCfromwholeblood:

1.1.firstpoolbloodofalldonorsintoacultureflaskthendividethebloodover2or450mltubes.

Thesetubescanbespindownat900g,for10minuteswithoutbrake.

1.2.Removetheserumandasmuchofthebuffycoat(whitecelllayer)aspossible.

1.3.ResuspendinaequalvolumeorexcessofS.A.G.M.solutionandwashthecellsfor5minutesat900gwithoutbrake.RemovetheS.A.G.Msolutionandanyremainingvisiblewhitecellfromthesurfaceoftheredbloodcells.

1.4.Repeatthewashasstatedin1.3.twicemore.

1.5.Afterthefinalwash,measuretheredbloodcellvolumeandresuspendinanequalvolumeof

S.A.G.M.anddividethe50%HTcellsuspensionover8-12sterile15mltubes.

1.6.Storeat4°C.

2.PreparationofuninfectedRBCforuseinGIA:

2.1.UsethesamebatchofRBCusedfortheculturingofparasitesinthelastfewdays.

2.2.MakeaRBCsuspensionof4%hematocritin2xCmed.

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D.2.3.Preparationofthetest

samplesUseProteinGcolumns.

1.*Dilute1partserumsamplein2partsbindingbuffer.

2.Leaveo/nat4°C.

3.Spinserumsamples10minutes2400g,andremovefat(whitering)ontop.

4.TransfersupernatantinacleantubeanduseforIgG-purification.

5.Washcolumnswith2xcolumnvolumeofbindingbuffer;pumpspeed=10rpm(~1.4ml/min).

6.*Applysamplesoncolumns;pumpspeed=10rpm(~1.4ml/min),collecttheflowthroughin50mltubeslabeledwiththesample-nameand“flowthrough”.

7.Washcolumnswith2xcolumnvolumeofbindingbuffer;pumpspeed=10rpm(~1.4ml/min),

8.*Washcolumnswith35-40xcolumnvolumeofPBS;pumpspeed=20rpm(~2.8ml/min),Collecttheflowthrough(totalof100mlincludingtheflowthroughofstep6and7),therestoftheflowthroughiswaiste.

9.*Takeaflowthroughsampletomeasureonthenanodrop(blank=PBS,samplehastobebelow

0.1mg/ml,otherwisecontinuewashingwithPBSuntilsampleisbelow0.1mg/ml).

10.Prepare50mltubeswith3mlof1MTRIS(pH9.0)(1tubepersample),labeledwithsamplename.

11.Washcolumnswithelutionbuffer;pumpspeed=10rpm(~1.4ml/min),collecttheflowthroughintheprepared50mltubesuntilatotalvolumeof30ml.

12.*Takethelastdropofflowthroughtomeasureonthenanodrop(blank=PBS,samplehastobebelow0.1mg/ml,otherwisecontinuewashingwithelutionbufferuntilsampleisbelow0.1mg/ml).

13.*MeasureyourcollectedflowthroughonthenanodropforanindicationoftheIgG-yield.

14.Washcolumnswith2xcolumnvolumeofPBS;pumpspeed=20rpm(~2.8ml/min).

15.Washcolumnswith1xcolumnvolumeofNaAz;pumpspeed=20rpm(~2.8ml/min).

16.Pour10ml70%EtOHontheAmiconUltraFiltertubesandleavethemfor5minutes.

17.PourofftheEtOHandletthefiltersdry.

18.Pour15mlplainRPMIonthefilters(tocleanouttheEtOH).

19.Spinfor10minutesat1900g.

20.FiltertheelutedIgG’sthrougha0.2µm-filterusingasyringe.

21.Pour15mloftheelutedIgG’sontheAmiconUltraFilterTubes.

22.*Spinfor30minutesat1900g(longerifthereismorethan2mloffluidleftabovethefilter).

23.*Collecttheflowthroughinanew50mltubelabeledwaiste1.

24.Pourtheremaining15mloftheelutedIgG’sontheAmiconUltraFilterTubes.

25.Spinfor30minutesat1900g(longerifthereismorethan2mloffluidleftabovethefilter).

26.Collecttheflowthroughinthewaiste1-tube.

27.WashthetubeusedtheelutedIgG’swith5mlofplainRPMIandpourovertotheAmiconUltraFilterTubes.

28.FilltheAmiconUltraFilterTubesupto15mlwithplainRPMI.

29.Spinfor30minutesat1900g(longerifthereismorethan2mloffluidleftabovethefilter).

30.Collecttheflowthroughinthewaiste1-tube.

31.FilltheAmiconUltraFilterTubesupto15mlwithplainRPMI.

32.Spinfor30minutesat1900g(longerifthereismorethan2mloffluidleftabovethefilter).

33.Collecttheflowthroughinthewaiste2-tube.

34.FilltheAmiconUltraFilterTubesupto15mlwithRPMI.

35.Spinfor30minutesat1900g(longerifthereismorethan2mloffluidleftabovethefilter).

36.Collecttheflowthroughinthewaiste2-tube.

37.TransfertheconcentratedIgGtoa1,8mlscrew-captube.

38.*Measurea1in10diluted(inRPMI)sampleonthenanodrop.

39.*DilutetheIgG’stothewantedconcentration,theconcentrationis2xtheend-concentrationwantedinGIA.

40.Storeat-20°CuntiluseinGIA.

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D.2.4.PreparationofGIAparasitesuspension

1.Counttheparasitaemiainasmear(seeD.2.2.1.3-D.2.2.1.19),makingsureallparasitesareinthelate-schizontstage.

2.Transfertheparasite-culturetoa50mltube.

3.Spinfor5minutesat900g.

4.Removesupernatant.

5.Diluteenoughoftheparasite-pelletin2xCmedfor0.2-0.3%parasitaemia.

6.AddRBC50%HTtogeta4%HTsuspension.

D.2.5.GIAset-up

Theassaycouldbeperformedwithanendvolumeof30µlor50µl.A30µlassayiscouldbeusedwhenyoudonothaveenoughIgGsample,orjusttosavesomeforfutureassays.

1.Use96-wellflat-bottomhalf-areacultureplateswithindividuallids.

2.*Pipette15-25µlIgG’sintriplicates(30-50µlifyoumakeaserialdilutionoftheIgG’s,thanalsopipette15-25µlofplainRPMIintheotherwellsforserialdilution).

3.*Pipette15-25µlpositiveIgG.

4.*Pipette15-25µlnegativeIgG.

5.*Pipette15-25µlplainRPMIfortheschizontcontrolswells,RBC-controlwellsandtothemonitorwells.

6.*Pipette15-25µl8mMEDTAfortheEDTA-control.

7.*Add15-25µlparasite-suspensiontoallwells,exceptRBC-controlwells.

8.*Add15-25µlRBC-suspensiontotheRBC-controlwells.

9.Puttheplatesinahumidifiedbox(wettissuesorwateratthebottomofthebox),andplacethelidonthebox,butdon’tcloseitcompletely.

10.Puttheboxeswiththeplatesinahumidifiedincubatorat37°C,containing5%CO2,5%O2,90%N2.

11.Incubatefor40–44hours.

12.Prepareasmearusingcontentsof3monitor-wellsforoneortwoplatesatT=0andafter

∼24hrsandstainwithGiemsatocheckparasite-stageandparasitemia.Finalhematocrite:2%

Finalparasitaemia:0.3±0.1%

D.2.6.GIAharvest

1.Prepareasmearusingcontentsof3monitor-wellsofeachplateatT=∼44hrsandstainwithGiemsatocheckparasite-stage andparasitemia. Harvestatschizont-stage.

2.*Fillnew96-wellflat-bottomcultureplateswith200µl/wellofcoldPBS(oneplateforeachassayplate).

3.MixthecontentsofthewellsintheassayplatethoroughlyusingamultichannelandtransferthecontentstothePBS-plate(inthesameplateformat).

4.Centrifugeplatesfor10minat1300xgat4°Cwithoutbrake.

5.Remove190µlofsupernatantwithoutdisturbingthepellet.

6.Freezetheplatesovernightat-20°C(oruntilreadyforpLDH-assay)tolyseerythrocytes.

D.2.7.pLDHassay

NOTE:NBTislightsensitive,soavoiddirectlightandkeepsolutionsandplateswithsubstrateindark(coverwithaluminiumfoil)

1.ThawtheplatesandLDH-bufferandwarmuptoroomtemperature(10mlLDH-buffer/plate).

2.DissolveNBTinLDH-bufferataconcentrationof2mg/10ml.Mixgentlyandkeepsubstrateindark.

3.Add50µlAPADstock(10mg/ml)toevery10mlsubstrate.

4.Add200µlDiaphorasestock(50units/ml)toevery10mlsubstrate.

5.Usesubstrateimmediately.

6.*Add100µlsubstrateperwelloftheharvestedplates.Oneplateeveryminute.

7.Coverwithaluminium-foilandplaceonaflatbedshakerat400rpmatroomtemparature.

8.Incubatefor30minutes.

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9.*MeasureODafter30minutesatwaveleangth655nm(A655).Oneplateeveryminute.

D.3.Plate-layout

Twoexamplesofplate-layouts,withfinalIgG-concentrations.

D.4.Remarkstotheprocedure

Ad.D.2.1.1Culturing3timesaweek:PreferablyonMondaymorning,Wednesdaymid-day,Fridayafternoon.

Ad.D.2.1.1.10.Giemsa-stockis5xconcentrated.AfreshGiemsa-stainismadebydiluting1partGiemsa-stockin4partsbufferpH7.2.

Ad.D.2.1.1.14.Useatissuefromthepaper-dispenser.Pressthetissueontotheslidewithoutwhipingtheslide.Repeatthis.Theslideshouldbedry.

Ad.D.2.1.1.19.Determinationofthedevelopmentalstageoftheparasite-culturecouldbedoneduringthecountingoftheparasites(D.2.2.1.16).

Ad.D.2.1.2.13.Useanewculture-flaskafteralanine-treatmentbecauseintheoldculture-flasktherearestillsomeparasitesleftthatarenon-synchronised.

Ad.D.2.3.1.E.g.10mlofserum+20mlbindingbuffer.

Ad.D.2.3.6.First100mlofflowthroughiscollected/sampleincasethepurificationdidn’twork.

ThanitmightbethatalltheIgG’sareinthispartoftheflowthrough.Ad.D.2.3.8.E.g.column-volumeis5ml,thanwashwithatleast175mlofPBS.

Ad.D.2.3.9.Nanodrop:OpenND-1000V3.2.1-software.Cleanthepedestalwithaquadest.Putonpedestaltheblank(=PBS).ChooseProteinA280.OK.Undersample-typechoose“BSA”.Reblank(withoutputtingnewblankonpedestal).Cleanpedestal.Putonsample,namesampleinsample-field.Measure.Cleanpedestaletc.Atendofmeasuringsamples:Showreport.Printandsavewindow.

Ad.D.2.3.12.Nanodrop:OpenND-1000V3.2.1-software.Cleanthepedestalwithaquadest.Putonpedestaltheblank(=PBS).ChooseProteinA280.OK.Undersample-typechoose“IgG”.Reblank(withoutputtingnewblankonpedestal).Cleanpedestal.Puton

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sample,namesampleinsample-field.Measure.Cleanpedestaletc.Atendofmeasuringsamples:Showreport.Printandsavewindow.

Ad.D.2.3.13.SamewayasAd.D.2.4.12.

Ad.D.2.3.22.IfthereisarelativehighconcentrationofIgGinyourelutedIgG-solution,itcouldbethatthefilterneedsmoretimetoletthefluidthrough.Thanmostofthetimesanextra10minutespincouldbesufficient.ItcouldalsobethattheIgGconcentrationisveryhigh.Thanit’sbettertotransferpartofittoanewAmiconUltraFilterTubeandstartover.

Ad.D.2.3.23.TheflowthroughfromtheAmiconUltraFiltertubesiscollectedincasethefiltersleak.Whentheyleak,theywellalsopasstheIgG’s.Bycollectingtheflowthrough,youcanalwaystryagainbyfilteringtheflowthroughagainonanewAmiconUltraFiltertube.

Ad.D.2.3.38.TheconcentrationisnowmuchhigherthanintheoriginalelutedIgGsolution,so

diluteyousample1in10(inRPMI)formeasurementonthenanodrop.Useofthenanodrop:seeAd.D.2.4.12.

Ad.D.2.3.39.Whenyouneedtodiluteyoursample2x,it’sbetternottodoitin1step.Mostofthetimesyouwilldiluteyoursampletoofar,andyouneedtoconcentrateitagain.Sofirstdiluteit1.5x.

Concentrations wanted(withtheerror allowed):HumanIgG:20mg/ml(18–22mg/ml)

RhesusIgG:12mg/ml(10.8–13.2mg/ml)

RabbitIgG:12mg/ml(10.8–13.2mg/ml)Ad.D.2.5.2-8.Seeexampleplate-layoutsinD.3.

Ad.D.2.6.2.PutthecoldPBSinthefridgethedaybeforeharvesting.PlatecanbefilledwithPBSthedaybefore,butthanplacetheplatesinthefridgetokeepthemcold.ThecoldPBSstopstheparasitesintheircycle.

Ad.D.2.7.6.Addsubstrateto1plateperminute.Wraptheplatewithaluminium-foilandputontheflatbedshaker.Thistoensureallplatesareincubatedexactlythesametime.

Ad.D.2.7.9.Measure1plateperminute.Thisagaintoensureallplatesareincubatedexactlythesametime.

E.Results

OD-valuesmeasuredbytheplatereadershouldbeexportedtoacsv-file.

%inhibitionscanbecalculatedusingthefollowingformula:

(A655IgGsample–A655RBCcontrol)

%inhibition=100%-x100%

(A655SZcontrol–A655RBCcontrol)