Immunoassaysareapowerfultechniquefordetectingandmeasuringantigensandantibodies.Immunoassayscanbeclassifiedthreewaysbasedonthestepsinvolved:

• antibodycapture
• antigencapture
• two-antibodysandwich

Manytypesofimmunoassayscanbeusedtodetectandquantitatebothantigensandantibodies,buttherearedifferencesintheAvidityrequirementsfortheantibodies,thesignalstrengthsofthelabels,andtheamountofbackgroundforeachofthesetypesofassays.Antibodycaptureassaysarethemostappropriateformeasuringthetiteroftheantiserayouhavegenerated.

ELISAProcedureforMeasuringSerumAntibodyTiter

Thisprocedurehastwoparts.Part1appliestoanydetectionprotocol.Part2describestwodifferentdetectionmethods.Tomeasureanantibodytiter,decideonthedetectionmethodfirst,thencompletebothParts1and2.

Part1:AntigenandAntibody

EquipmentNeeded

• Microtiterplates
• Pipettor,1mLadjustable
• Eight-channelpipettor(optional)
• UV/vismicrotiterplatereader
• Pipettetips

SolutionstoPreparefortheELISAProcedure

• Sodiumcarbonate(Na₂CO₃)buffer,50mM,pH9.6with0.02%NaN₃
• PBS-T(PBScontaining0.05%Tween-20)
• 3%BSAinPBS-T
• Sodiumacetate(NaOAc),0.1M

ELISAProcedure

1.Choosethedetectionmethod.Note:IfyouchoosehorserADIshperoxidasedetection,omitthesodiumazidefromthebuffers.

2.Preparebuffersolutions.

3.Prepareasolutionofthepeptide,proteinorpeptide/conjugate(10µg/mLwithrespecttothepeptideorprotein)insodiumcarbonatebuffer.

4.Pipette200µLofeitherpeptide,peptidecarrierconjugate,orsodiumcarbonatebuffer(forcontrols)intheindividualwellsofthemicrotiterplate.

Table3.ELISATemplate

5.Covertheplateandincubateforthreehoursatroomtemperatureorovernightat2-6°C.

6.RemoveunboundantigenbywashingthreetimeswiththePBS-Tbuffer.Asquirtbottleisahandywaytoaddthebuffertothewells.Removetheantigensolutionandwashesbyinvertingtheplatequicklyandtappingthebottomonpapertowelstoremoveanydrops.Thisensuresthatthereisnocrosscontaminationordilution.

7.Blocktheremainingadsorptionsitesbyadding300µLof3%BSA/PBS-Ttoeachwell.Incubateforonehouratroomtemperature.

8.WashtheplatestwicewithPBS-T.

9.Preparea1:50dilutionoftheserabypipetting60µLoftheserainto2940µLofPBS-T.Usemicrocentrifugetubeswithflip-topcaps.Storealldilutionsonice.

10.PrepareserialdilutionsusingthedilutionscheduleinTable4.

11.Vortexthetubeswell.Storealldilutionsonice.

12.Pipette200µLoftheantiseradilutionsintothewells.

13.Incubateforthreehoursatroomtemperatureorovernightat2-6°C.

14.WashwithPBS-Ttoremovetheunboundantibodies.

Thenextstep(Part2)isdetectionoftheamountofantibodiesboundtotheplate.Youcanchooseeitheroftheproceduresdescribed.

NotesonELISAProcedures

1.Bindingofpeptidestomicrotiterplatescanbeproblematicinthatthepeptidemaynotbindormaybindinaconfigurationthatmasksordistortstheepitopessothattheyarenotrecognizedbytheantibody.Foralternativebindingmethods,seeWisdom(1994),Chapter6.

2.Itisimportanttocheckthespecificityofyouranti-peptideantiserumtowardstheproteinofinterest.Thiscanbedonebyanumberofmethods,includingimmunoblotting(usingdenaturingandnativegels)andimmunocytochemistry.Forprotocolsofboththesemethods,seeHarlowandLane(1988).

Part2:AntibodyDetectionandMeasurement

DetectionUsingAlkalinePhosphatase

ReagentsforAlkalinePhosphataseDetection

• Goatanti-speciesIgGwholemolecule/alkalinephosphataseconjugatethatisappropriateforthesourceofyourantiserum.

  Forexample,iftheantibodieswereraisedinarabbit,usegoatanti-rabbitIgGwholemolecule/alkalinephosphataseconjugate

• p-Nitrophenylphosphate
• Sodiumcarbonatebuffer,0.05MpH9.8,containing1mMmagnesiumchloride(MgCl₂)
• Sodiumhydroxide(NaOH),2M

ProcedureforAlkalinePhosphataseDetection

1.PrepareadilutionofthegoatIgG/alkalinephosphataseconjugate(usually1:1000orgreater)thatisappropriatetothesourceofyourantibodiesinPBS-T.Pipette200µLofthissolutionintoeachofthewellsandincubateforthreehoursat37°Corovernightat2-6°C.

2.RemovetheunboundconjugatebywashingwithPBS-T.

3.Add200µLofa10mg/mLso,lutionofp-nitrophenylphosphatein0.05Msodiumcarbonatebuffer(pH9.8,containing1mMMgCl₂),slowlyatroomtemperatureuntilayellowcolordevelops,approximately10-60minutes.

4.Add50µLof2MNaOHtostopthereaction.

5.Measuretheabsorbanceofeachwellat405nm.

DetectionUsingHorseradishPeroxidase

Note:Ifhorseradishperoxidaseisused,donotincludesodiumazideinthewashbuffer.

ReagentsforHorseradishPeroxidaseDetection

1.Goatanti-speciesIgGwholemolecule/horseradishperoxidaseconjugatethatisappropriateforthesourceofyourantiserum.

Forexample,iftheantibodieswereraisedinarabbit,usegoatanti-rabbitIgGwholemolecule/horseradishperoxidaseconjugate.

2.3",3",5",5"-Tetramethylbenzidine(TMB)

3.Hydrogenperoxide,30%solution

4.Sulfuricacid(H₂SO₄),1M

ProcedureforHorseradishPeroxidaseDetection

1.PrepareadilutionofthegoatIgG/horseradishperoxidaseconjugate(usually1:1000orgreater)thatisappropriatetothesourceofyourantibodiesinPBS-T(themanufactureroftheantibodiesshouldprovideyouwiththisinformation).Pipette200µLofthissolutionintoeachofthewellsandincubateforthreehoursat37°Corovernightat2-6°C.

2.RemovetheunboundconjugatebywashingtheplatewithPBS-T.

3.Prepareasolutionof0.1mgofTMBin100µLdimethylsulfoxide.Add9.9mLof0.1MsodiumacetateandfilterthroughaWhatmanNo.1filter.Addenoughhydrogenperoxidetogiveafinalconcentrationof0.01%.

4.Add100µLofthesubstratesolutiontoeachmicrotiterwell.

5.Covertheplateandincubateatroomtemperaturefor10-60minutes.Ablueproductwillform.

6.Thereactionmaybestoppedbytheadditionof50µLof1MH₂SO₄.

7.Readabsorbanceat450nmforthestoppedproductandat650nmfortheunstoppedreaction.

HowtoCalculatetheAntibodyTiteroftheSera

1.Plotabsorbancevs.antiserumdilutionusingthemeanandrangeorstandarddeviationforeachduplicateortriplicateset.

2.Estimatetheinflectionpointofthepost-immunegraph.

3.Interpolatethetiterbydrawingalinedowntothex-axis.

AnexampleofagraphusedtodeterminethetiterofanantibodyisshowninFigure22.Thetiterwasestimatedtobe1in2400.

Figure22.Antibodytitergraph(X=dilutioncorrespondingtoAbtiter)

FurtherMethodsforEvaluatingAnti-peptideAntibodies

Iftheantibodiesaretobeusedasareagenttostudyaspecificprotein,itisimportanttolearnwhethertheyrecognizethenativeprotein.ThiscanbedoneusingtheaboveELISAprocedure.Usetheproteininplaceofthepeptideorpeptideconjugate.Additionalinformationaboutthebindingstrengthoftheantibodiescanbeobtainedbyacompetitiveassayusingthesyntheticpeptideinconjunctionwiththenativeprotein.Forfurtherdetails,seeVanRegenmortel(1988).