human acute phase panel 1 mix and match subpanel · legendplex™ human acute phase panel 1 mix and...
TRANSCRIPT
LEGENDplex™ Kits are manufactured by BioLegend 8999 BioLegend WaySan Diego, CA 92121Tel: 1.858.768.5800Tel: US & Canada Toll-Free: 1.877.Bio-Legend (1.877.246.5343)Fax: 1.877.455.9587Email: [email protected]
For a complete list of world-wide BioLegend offices and distributors, please visit our website at: biolegend.com
Enabling Legendary Discovery™
750000670_V01
LEGENDplex™Mul�-Analyte Flow Assay Kit
Enabling Legendary Discovery™
Human Acute Phase Panel 1 Mix and Match Subpanel
Please read the entire manual before running the assay.
BioLegend.com
For Research Purposes Only. Not for use in diagnostic or therapeutic procedures. Purchase does not include or carry the right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of BioLegend is strictly prohibited.
It is highly recommended that this manual be read in its entirety before using this product. Do not use this kit beyond the expiration date.
biolegend.com1
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Table of Contents Page
Chapter 1: KIT DESCRIPTION..................................................
Introduction……………………………………………..........................
PrincipleoftheAssay……………………....……………....….…......
BeadsUsage...........................................………..……………...
StorageInformation…………………………………….......…..........
MaterialsSupplied………………….....……………….................…
MaterialstobeProvidedbytheEnd-User……...........……...
Precautions.................................……………………................
Chapter 2: ASSAY PREPARATION..............................................
SampleCollectionandHandling…………………………............
ReagentPreparation………………..………………………...............
StandardPreparation.........................................................
SampleDilution……...........……............................................
Chapter 3: ASSAY PROCEDURE..................................................
PerformingtheAssayUsingaFilterPlate……………….........
PerformingtheAssayUsingaV-bottomPlate……….........
Chapter 4: FLOW CYTOMETER SETUP.......................................
Chapter 5: DATA ACQUISITION AND ANALYSIS.........................
DataAcquisition..................................................................
Data Analysis......................................................................
Chapter 6: ASSAY CHARACTERIZATION....................................
RepresentativeStandardCurve.………………………………........
AssaySensitivity...……………………………………………………..…..
Cross-Reactivity........................………………………………………
Accuracy.............................................................................
LinearityofDilution………………………………………………..........
3
3
3
4
6
6
8
9
10
10
10
11
12
13
13
16
19
19
19
20
21
21
21
21
22
23
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
2
Intra-AssayPrecision……………………………………...................
Inter-AssayPrecision……………………………………...................
BiologicalSamples…………………………………………….………....
TROUBLESHOOTING..........................…………………………….....…....
PLATE MAP...........................……………………………………………………..
24
24
25
27
33
biolegend.com3
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Chapter 1: KIT DESCRIPTIONIntroduction
Theacutephaseresponseisacomplexsystemicreactiontriggeredbytheonsetofvariouscriticalconditionsincludingtrauma,infection,tissuedamage,inflammation,stress,orneoplasia.Themostimportantchangeofthisresponseisthehighlyincreasedproductionofalargefamilyofproteinsfromtheliver,collectivelyknownasacutephaseproteins.Consideredasapartofinnateimmunity,acutephaseproteinsareresponsibleformanysystemiceffectssuchasanti-proteaseactivities,leukocytosis,complementcascade,increasedcortisol,andmanyothers.Measuringandmonitoringacutephaseproteinsprovideresearchvalueinunderstandingthemechanisms,therapeutics,andprognosis of many diseases.
The LEGENDplexTMHumanAcutePhasePanel1(8-plex)containsfluorescence-encodedbeadssuitableforuseoncommonflowcytometers.Itallowsforthesimultaneousquantificationof8keyacute phase molecules includingα2-microglobulin,α1-AcidGlycoprotein(α1-AGP),Haptoglobin,α1-antitrypsin,Ceruloplasmin,Fibrinogen,Prothrombin,andSerumAmyloidPComponent(SAP).ThisassaypanelprovideshigherdetectionsensitivityandbroaderdynamicrangethantraditionalELISAmethods.Thepanelhasbeenvalidatedforuseoncellculturesupernatant,serum,andplasmasamples.
The Human Acute PhasePanel1isdesignedtoallowflexiblecustomizationwithinthepanel.Pleasevisitwww.biolegend.com/legendplex for more informationonpaneldesignandhowtomixandmatchwithinthepanel.
This assay is for research use only
Principle of the Assay
BioLegend’s LEGENDplexTM assays are bead-based immunoassays that use the samebasicprincipleassandwichimmunoassays.
Beadsaredifferentiatedbysizeandinternalfluorescenceintensities.Thesur-faceofeachbeadsetisfirstconjugatedwithspecificantibodies,andthenusedascapturebeadsforthatparticularanalyte.Whenaselectedpanelofcapturebeadsaremixedandincubatedwithasamplecontainingtargetanalytes,eachanalytewillbindtoitsspecificcapturebeads.Afterwashing,abiotinylatedde-tectionantibodycocktailisadded,andeachdetectionantibodyinthecocktailwillbindtoitsspecificanalyteboundonthecapturebeads,thusformingcap-turebead-analyte-detectionantibodysandwiches.Streptavidin-phycoerythrin(SA-PE)issubsequentlyadded,whichwillbindtothebiotinylateddetectionantibodies,providingfluorescentsignalintensitiesinproportiontotheamountof bound analytes.
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
4
Sincethebeadsaredifferentiatedbysizeandinternalfluorescenceintensityonaflowcytometer,analyte-specificpopulationscanbesegregatedandPEfluorescentsignalquantified.Theconcentrationofaparticularanalyteisdeter-mined using a standard curve generated in the same assay.
Beads Usage
The Human Acute Phase Panel 1usestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescat-ter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.TheinternaldyecanbedetectedusingeithertheFL3,FL4,orAPCchannels,dependingonthetypeofflowcytometerused.ThesmallerABeadsconsistsof6beadpopulationsandthelargerBBeadsconsistsof7beadpopulations(Figure2-3).FourPopulationsof each set of beads are used for this panel (indicated as Beads ID in Table 1).
Usingatotalof8beadpopulationsdistinguishedbysizeandinternalfluores-centdye,theHuman Acute Phase Panel 1allowssimultaneousdetectionof8proteinsinasinglesample.Eachanalyteisassociatedwithaparticularbeadsetas indicated (Figures 2-3 and Table 1).
Figure 1. Beads Differentiated by Size
Beads A = smaller beads
Beads B = larger beads
Figure 2. Beads A Classification by FL4
A5 A7 A8
A4
A6
A10
biolegend.com5
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match SubpanelFigure 3. Beads B Classification by FL4
ForBeadsusageinthefullpanel,pleaserefertoTable1below.
Table 1. Panel Targets and Bead ID*
Target Bead ID
Human Acute Phase Panel 1 (8-plex) Mix and
MatchTop Standard
ConcentrationsCat. # 740999 or 741000
α2-macroglobulin A4 √The top standard
concentrationofeachtarget
mayvaryandmaysubject
to change from lot to lot.
Pleaserefertothelot-specific
CertificateofAnalysis
for this
information.
α1-AGP A5 √
Haptoglobin A7 √
α1-antitrypsin A10 √
Ceruloplasmin B3 √
Fibrinogen B4 √
Prothrombin B5 √
SAP B7 √
*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.
When entering analyte and bead ID infomation during the gating step, always enter in the sequential order of the bead ID (e.g, A4, A5... B3, B4...). Please refer to the LEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelpfor details (www.biolegend.com/legendplex).
B4 B5
B6 B7
B3
B9
B2
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
6
Storage Information
Recommendedstorageforalloriginalkitcomponentsisbetween2°Cand8°C.DONOTFREEZEPre-mixedBeads,DetectionAntibodiesorSA-PE.
• Oncethestandardshavebeensufficientlyreconstituted,immediatelytransfercontentsintopolypropylenevials.DONOTSTORERECONSTITUT-ED STANDARDS IN GLASS VIALS.
• Uponreconstitution,leftovertopstandardshouldbestoredat≤-70°Cforusewithinonemonth.Avoidmultiple(>2)freeze-thawcycles.Discardanyleftoverdilutedstandards.
Materials Supplied
The LEGENDplexTMkitcontainsreagentsfor100tests,listedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.
TheBufferSetcontainsSetupBeads,allBuffers,PlateSealers,SA-PEandDataAnalysisSoftwareDongle.AmanualisalsoprovidedforeachMixandMatchsubpanel.
Table 2: The LEGENDplexTM Kit Human Acute Phase Panel 1 Mix & Match Sub-panel Kit Components
Kit Components Quantity Volume Cat #Capture Beads (see below Table 3 for more information) Varies Varies Varies
HumanAcutePhasePanel1DetectionAntibodies 1bottle 3.5 mL 741001
Human Acute Phase Panel 1 Standard 1 vial Lyophilized 741002
LEGENDplex™BufferSetN(seebelowTable4formoreinformation)
1 -- 741003
Filter Plate* or V-bottomPlate** 1 plate -- 740377* or
740379**
Human Acute Phase Panel 1 Mix/Match Sub Manual 1 -- 750000670
*Forkitwithfilterplate.**ForkitwithV-bottomplate.Onlyoneplateispro-videdforeachkit.
biolegend.com7
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Table 3: Capture Beads for Mix and Match Subpanels***
Kit Components Quantity Volume Cat.#Humanα2-macroglobulinCaptureBeadA4,13X 1 vial 270 µL 741004
Humanα1-AGPCaptureBeadA5,13X 1 vial 270 µL 741005
HumanHaptoglobinCaptureBeadA7,13X 1 vial 270 µL 741006
Humanα1-antitrypsinCaptureBeadA10,13X 1 vial 270 µL 741007
HumanCeruloplasminCaptureBeadB3,13X 1 vial 270 µL 741008
HumanFibrinogenCaptureBeadB4,13X 1 vial 270 µL 741009
HumanProthrombinCaptureBeadB5,13X 1 vial 270 µL 741010
HumanSAPCaptureBead,B7,13X 1 vial 270 µL 741011 *** Please refer to Panel Targets and Bead ID (Table 1, page 5), toseewhichcapture beads are selected in each panel.
Table 4: LEGENDplex™ Buffer Set N (Cat# 741003 )
Components Quantity Volume Part #Setup Beads 1: FITC Beads 1 vial 1 mL 77840Setup Beads 2: PE Beads 1 vial 1 mL 77842SetupBeads3:RawBeads 1 vial 2 mL 77844LEGENDplexTM SA-PE 1bottle 3.5 mL 77743LEGENDplexTMAssayBuffer 3bottles 75 mL 77562Lyophilized Standard ReconstitutionBuffer 1 vial 1 mL 75241
LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564DataAnalysisSoftwareDongle 1 -- 21217Plate Sealers 4 sheets -- 78101NoplateisincludedinBufferSetN.Plateneedstobeorderedseparately.Please order the correct type of plate based on the preferred assay protocol (Cat# 740377 or 740378 forFilterPlateandCat#740379forV-bottomPlate).
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
8
Materials to be Provided by the End-User
• Aflowcytometerequippedwithtwolasers(e.g.,a488nmbluelaseror532nmgreenlaseranda633-635nmredlaser)capableofdistinguishing575 nm and 660 nm oraflowcytometerequippedwithonelaser(e.g.,488nmbluelaser)capableofdistinguishing575nmand670nm.
Partial list of compatible flow cytometers:
Flow Cytometer
Reporter Channel
ReporterEmission
Classification Channel
Channel Emission
Compen-sation
needed?
BD FACSCaliburTM FL2 575 nm FL4 660 nm No*
BD AccuriTM C6 FL2 585 nm FL4 675 nm No*
BD FACSCantoTM,BD FACSCantoTM II PE 575 nm APC 660 nm No*
BDTMLSR,LSRIIBD LSRFortessaTM PE 575 nm APC 660 nm No*
GalliosTM PE 575 nm APC 660 nm No*
CytoFLEX PE 585 nm APC 660 nm No*
NovoCyte PE 572 nm APC 660 nm No*
AttuneTM NxT PE 574 nm APC 670 nm No**Compensation is not required for the specified flow cytometers when set up properly.
Forsettingupvariousflowcytometers,pleasevisit:www.biolegend.com/legendplex andclickontheInstrument Setup tab.
• Multichannelpipettescapableofdispensing5μLto200μL
• Reagentreservoirsformultichannelpipette
• Polypropylene microfuge tubes (1.5 mL)
• MicroFACStubes,1.1mL(iftheflowcytometerdoesnotcontainanautos-ampler)
• Laboratory vortex mixer
• Sonicatorbath(e.g.,BransonUltrasonicCleanermodel#B200,orequiva-lent)
• Aluminum foil
• Absorbentpadsorpapertowels
• Plateshaker(e.g.,Lab-LineInstrumentsmodel#4625,orequivalent)
• Tabletopcentrifuges(e.g.,Eppendorfcentrifuge5415C,orequivalent)
biolegend.com9
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
If the assay is performed in a filter plate:
•Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,cat#MSVMHTS00orequivalent).Instructionsonhowtousethevacuummanifoldcanbefoundatthesupplier’swebsite.
•Avacuumsource(minivacuumpumporlinevacuum,e.g.,MilliporeVacuumPump,catalog#WP6111560,orequivalent)
• Ifneeded,additionalFilterplatescanbeorderedfromBioLegend(Cat#740377 or 740378).
If the assay is performed in a V-bottom plate:
• Centrifugewithaswingingbucketadaptorformicrotiterplates(e.g.,Beck-man Coulter AllegraTM6RCentrifugewithMICROPLUSCARRIERadaptorforGH3.8 and JS4.3 Rotors) .
• Ifneeded,additionalV-bottomplatescanbeorderedfromBioLegend(Cat# 740379).
Precautions
• All blood components and biological materials should be handled as potentiallyhazardous.FollowuniversalprecautionsasestablishedbytheCenterforDiseaseControlandPreventionandbytheOccupationalSafetyandHealthAdministrationwhenhandlinganddisposingofinfectiousagents.
• Sodiumazidehasbeenaddedtosomereagentsasapreservative.Al-thoughtheconcentrationsarelow,sodiumazidemayreactwithleadandcopperplumbingtoformhighlyexplosivemetalazides.Ondisposal,flushwithalargevolumeofwatertopreventazidebuild-up.
• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.
• Donotusethiskitbeyonditsexpirationdate.
• SA-PEandbeadsarelight-sensitive.Minimizelightexposure.
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
10
Chapter 2: ASSAY PREPARATION
Sample Collection and Handling
Preparation of Serum Samples:
• Allowthebloodtoclotforatleast30minutesandcentrifugefor20min-utesat1,000xg.
• Remove serum and assay immediately or aliquot and store samples at ≤-20°C.Avoidmultiple(>2)freeze/thawcycles.
• Whenusingfrozensamples,itisrecommendedthatsamplesbethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.
Preparation of Plasma Samples:
• Plasmacollectionshouldbecollectedusingananti-coagulant(e.g.,EDTA,Heparin,Citrate).Centrifugefor20minutesat1,000xgwithin30minutesofbloodcollection.
• Removeplasmaandassayimmediately,oraliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.
• Whenusingfrozensamples,itisrecommendedthatsamplesbethawedcompletely,mixedwellandcentrifugedtoremoveparticulates.
Preparation of Cell Culture Supernatant:
• Centrifuge the sample to remove debris and assay immediately. If not pos-sible,aliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.
Reagent Preparation
Preparation of Antibody-Immobilized Beads
1. Theindividualbeads(13X)needtobecombinedwithoneanotheranddilutedwithAssayBuffertocreatea1Xworkingsolutionofbeadspriorto use.
2. Sonicate each bead vial for 1 minute in a sonicator bath and then vortex for 30 seconds to completely resuspend the beads.
3. Calculateandpreparea1Xbeadsworkingsolutionbasedonthedesirednumberofreactionsandplex-sizeofyourassay(i.e.thenumberofindi-vidualbeadvials)followingthestepsdescribedbelow.
A. Total volume (µL) = 30x(numberofreactions)
B.Volumeneededfromeach13Xbeadsvial(µL)=2.3 x (number of
biolegend.com11
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
reactions)
C.AssayBufferneeded(µL)=A–Bx(numberofindividualbeadsvialsto be mixed)
Note:calculationsfortotalvolumeincludea20%excesstoaccountforanylossduringpipetting.
Example: to prepare 50 reactions for a 5-plex assay
A. Total volume (µL) = 30 x 50 = 1500 µL
B. Volume per beads vial needed (µL) = 2.3 x 50 = 115 µL
C.AssayBufferneeded(µL)=A–Bx(numberofindividualbeadsvials)=1500–(115x5)=925µL
Combine115µLofeachbeadsvial(5vials)with925µLofassaybuffertogetthedesiredfinalvolumeof1500µLof1Xworkingsolutionofbeads.
4. Sonicatepre-mixedBeadsbottlefor1minuteinasonicatorbathandthenvortexfor30secondspriortouse.Ifnosonicatorbathisavailable,increasethevortexingtimeto1minutetocompletelyresuspendthebeads.
Preparation of Wash Buffer
• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.
• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.
Standard Preparation
1. Priortouse,reconstitutethelyophilizedHumanAcute Phase Panel 1 Stan-dardwith250μLLyophilizedStandardReconstitutionBuffer
2. Mixandallowthevialtositatroomtemperaturefor10minutes,andthentransfer the standard to an appropriately labeled polypropylene microcen-trifugetube.ThiswillbeusedasthetopstandardC7.
Note: The top standard concentrations of analytes in this panel were set at various concentrations, but may be subject to change from lot to lot (see lot-specific Certificate of Analysis provided in the kit box for details).
3. Label6polypropylenemicrocentrifugetubesasC6,C5,C4,C3,C2andC1,respectively.
4. Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthe top standard by transferring 25 µL of the top standard C7 to the C6
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
12
tubeandmixwell.ThiswillbetheC6standard.
5. Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2and C1 standards (see the table below using the top standard at 10,000 pg/mL as an example).AssayBufferwillbeusedasthe0pg/mLstandard(C0).
Tube/Standard ID
Serial Dilution
Assay Buffer to add (µL)
Standard to add
Final Conc. (pg/mL)
C7 -- -- -- 10,000
C6 1:4 75 25 µL of C7 2,500
C5 1:16 75 25 µL of C6 625
C4 1:64 75 25 µL of C5 156.25
C3 1:256 75 25 µL of C4 39.01
C2 1:1024 75 25 µL of C3 9.77
C1 1:4096 75 25 µL of C2 2.44
C0 -- 75 -- 0
Sample Dilution
• Serumorplasmasamplesmustbediluted20,000-foldwithAssayBufferasdescribedinthetabelbelow.
Sample 1st Diluton(1:200)
2ndDilution(1 : 100) FinalDilutionFold
Serum,plasma
2 µL + 398 µLAssayBuffer
2µL1stDilution+198µLAssayBuffer 20,000
• Adding serum or plasma samples without dilution will result in low assay accuracy and possibly, clogging of the filter plate.
• Forcellculturesupernatantsamples,thelevelsofanalytecanvarygreatlyfrom sample to sample. To test cell culture supernatant samples,a prelimi-naryexperimentmayberequiredtodeterminetheappropriatedilutionfactor.Iffurtherdilutionisdesired,dilutionshouldbedonewithcorre-spondingfreshcellculturemediumorAssayBufferasadiluenttoensureaccurate measurement.
biolegend.com13
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Chapter 3: ASSAY PROCEDURE
The LEGENDplexTM assaycanbeperformedinafilterplate,orinaV-bottomplate.
Performing the Assay Using a Filter Plate
• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.
• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationsteps,sothatthebottomoftheplatedoesnottouchanysurface.Touchingasurfacemaycauseleakage.
• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.
• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.
• Standards and samples should be run in duplicate and arranged on the plate in a vertical configuration convenient for data acquisition and analysis (as shown in attached PLATE MAP, page 33). Be sure to load standards in the first two columns. If an automation device is used for reading, the orientation and reading sequence should be carefully planned.
1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeachwellandletitsitfor1minuteatroomtemperature.Toremovetheexcessvolume,placetheplateonthevacuummanifoldandapplyvacuum.Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypressingtheplateonastackofcleanpapertowels.Placetheplateontopof the inverted plate cover.
2. loadtheplateasshowninthetablebelow(intheorderfromlefttoright)For measuring cell culture supernatant samples:
Cell Culture Medium orAssayBuffer Standard Sample*
StandardWells 25 µL 25 µL --Samplewells 25 µL -- 25 µL
For measuring serum or plasma samples:AssayBuffer Standard Sample*
StandardWells 25 µL 25 µL ---
Samplewells 25 µL --- 25 µL *See Sample Dilution on page 12
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
14
Assay Procedure Summary for Filter PlateAdd 100 μL 1X Wash Bu�er to �lter plate wells
Vacuum to remove excess bu�er
Incubate 2 hours, RT, shaking
Capture beads
Biotinylated Detection Antibody
Analytes
Without washing, add 25 μL SA-PEIncubate 30 min, RT, shaking
Wash 2 times using vacuum �ltration unitAdd 25 μL Detection AntibodiesIncubate 1 hr, RT, shaking
Wash 2 times using vacuum �ltration unitAdd 150 μL of 1x Wash Bu�er Read on a �ow cytometer
Add to the plate:25 μL Assay Bu�er to all wells25 μL diluted standard to standard wells or 25 μL sample to sample wells25 μL pre-mixed beads to all wells
BA
C
A B C
A B C
biolegend.com15
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel3. Vortexmixedbeadsbottlefor30seconds.Add25μLofmixedbeadsto
eachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringadditionofthebeads,shakemixedbeadsbottleintermit-tentlytoavoidbeadsettling).
4. Sealtheplatewithaplatesealer.To avoid plate leaking, do not apply posi-tive pressure to the sealer when sealing the plate.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshaker,secureitwitharubberbandandshakeatapproximate500rpm for 2 hours at room temperature.
5. Do not invert the plate! Place the plate on the vacuum manifold and apply vacuumasbeforeinStep1.Add200µLof1XWashBuffertoeachwell.RemoveWashBufferbyvacuumfiltration.BlotexcessWashBufferfromthebottomoftheplatewithanabsorbentpadorpapertowels. Repeat this washingsteponcemore.
6. Add25µLofDetectionAntibodiestoeachwell.
7. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximately500rpmfor1houratroomtemperature.
8. Do not vacuum!Add25µLofSA-PEtoeachwelldirectly.
9. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximate500rpmfor30minutesatroomtemperature.
10. Repeat step 5 above.
11. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplateshakerfor1minute.
12. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note: Prolonged sample storage can lead to reduced signal).
Iftheflowcytometerisequippedwithanautosampler,readtheplatedi-rectly using the autosampler. Please be sure to program the autosampler to resuspend beads in the well immediately before taking samples. The probe height may need to be adjusted when using an autosampler.
Ifanautosamplerisnotavailable,thesamplescanbetransferredfromthefilterplateto micro FACS (or FACS) tubes and read manually.
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
16
Performing the Assay Using a V-bottom Plate
• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.
• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.
• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.
• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanalysis(asshowninattachedPLATEMAP,page33).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,theori-entationandreadingsequenceshouldbecarefullyplanned.
1. loadtheplateasshowninthetablebelow(intheorderfromlefttoright)For measuring cell culture supernatant samples:
Cell Culture Medium orAssayBuffer Standard Sample*
StandardWells 25 µL 25 µL --Samplewells 25 µL -- 25 µL
For measuring serum or plasma samples:
AssayBuffer Standard Sample*
StandardWells 25 µL 25 µL ---
Samplewells 25 µL --- 25 µL *See Sample Dilution on page 12
2. Vortexmixedbeadsfor30seconds.Add25μLofmixedbeadstoeachwell.Thetotalvolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringbeadsaddition,shakemixedbeadsbottleintermittentlytoavoidbeadsettling).
3. Sealtheplatewithaplatesealer.Covertheentireplatewithaluminumfoil to protect the plate from light. Shakeat800rpmonaplateshakerfor2 hours at room temperature (Depending on the shaker, the speed may need to be adjusted. The optimal speed is one that is high enough to keep beads in suspension during incubation, but not too high that it may cause sample to spill from the wells).
4. Centrifugetheplateat1050rpm(~250g)for5minutes,usingaswingingbucketrotor(G.H3.8)withmicroplateadaptor(Please refer to Materials to be Provided by the End-User, page 8). Donotuseexcessivecentrifugationspeedasitmaymakeithardertoresuspendbeadsinlatersteps.Make sure the timer of the centrifuge works properly and standby to make sure
biolegend.com17
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanelthe centrifuge reaches preset speed.
5. Immediatelyaftercentrifugation,dumpthesupernatantintoabiohazardwastecontainerbyquicklyinvertingandflickingtheplatein one continu-ous and forceful motion.Thebeadspelletmayormaynotbevisibleafterdumping the supernatant. Loss of beads should not be a concern as the beadswillstayinthetipofthewellnicely.Blottheplateonastackofcleanpapertowelanddraintheremainingliquidfromthewellasmuchaspos-sible. Be careful not to disturb the bead pellet.
Alternatively,removalofthesupernatantmaybecompletedusingamultichannelpipettesetat75µL. Try to remove as much liquid as possible withoutremovinganybeads. Be sure to changepipettetipsbetweeneachroworcolumn.
6. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Asecondwashisoptional,butmayhelpreducebackground.
7. Add25µLofDetectionAntibodiestoeachwell.
8. Sealtheplatewithanewplatesealer.Covertheentireplatewithalumi-num foil to protect the plate from light. Shakeat800rpmonaplateshakerfor 1 hour at room temperature.
9. Do not wash the plate!Add25µLofSA-PEtoeachwelldirectly.
10. Sealtheplatewithanewplatesealer.Wraptheentireplatewithaluminumfoilandshaketheplateonaplateshakeratapproximate800rpmfor30minutes at room temperature.
11. Repeatstep4,and5.
12. (Thiswashingstepisoptionalbuthelpstoreducethebackground.)Washthe plate by dispensing 200 μLof1XWashBufferintoeachwellandincu-bate for one minute. Repeat step 4 and 5 above.
13. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsbypipet-ting.
14. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note: Prolonged sample storage can lead to reduced signal).
Iftheflowcytometerisequippedwithanautosampler,thesamplescanberead directly. Please be sure to program the autosampler to resuspend beads in the well immediately before taking samples. The probe height may need to be adjusted when using an autosampler.
Ifanautosamplerisnotavailable,thesamplescanbetransferredfromtheplate to micro FACS (or FACS) tubes and read manually.
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
18
Assay Procedure Summary for V-bottom Plate
Incubate 2 hours, RT, shaking
Capture beads
Biotinylated Detection Antibody
Analytes
Without washing, add 25 μL SA-PEIncubate 30 min, RT, shaking
Spin down beads, remove supernatant Wash 1 time Add 25 μL Detection AntibodiesIncubate 1 hr, RT, shaking
Spin down beads, remove supernatant Wash 1 time (optional)Add 150 µL of 1x Wash Bu�er Read on a �ow cytometer
Add to the plate:25 μL Assay Bu�er to sample wells25 μL diluted standard to standard wells or 25 μL sample to sample wells25 μL mixed beads to all wells
BA
C
A B C
A B C
biolegend.com19
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Chapter 4: FLOW CYTOMETER SETUP
Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.
Thesetupinstructionshavebeenremovedfromthismanualanduploadedontoourwebsitetosavepaper.
Toaccessthesetupinstructions,pleasevisit:www.biolegend.com/legendplex andclickontheInstrument Setup tab.
Chapter 5: DATA ACQUISITION AND ANALYSIS
Data Acquisition
1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.
2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetup Guide).
3. Vortex each sample for 5 seconds before analysis.
4. Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire2,100beadsfora7-plexassayor3,000beadsfor a 13-plex assay). Do not set to acquire total events as samples may containlargeamountsofdebris.Instead,createalargegatetoincludebothBeads A and Beads B (gate A+B) and set to acquire the number of events in gateA+B.Thiswillexludemajorityofthedebris.
Note:Donotacquiretoofewortoomanybeads.ToofewbeadsacquiredmayresultinhighCVsandtoomanybeadsacquiredmayresultinslowdata analysis later.
5. Read samples.
Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforerecordingorswitchingtoacquisi-tionmode.
To simplify data analysis using the LEGENDplexTMDataAnalysisSoftware,readsamplesinthesameorderasshownonthePLATEMAPattachedattheendofthemanual.Foranin-plateassay,readcolumnbycolumn(A1,B1,C1...A2,B2,C2...).
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
20
Whennamingdatafiles,trytousesimplenameswithaconsecutivenum-beringforeasydataanalysis(e.g.forstandards,C0.001,C0.002,C1.003,C1.004,C2.005,C2.006,C3.007,C3.008,...C7.015,C7.016;forsamples,S1.017,S1.018,S2.019,S2.020,S3.021,S3.022…)
StoreallFCSfilesinthesamefolderforeachassay.Ifrunningmultipleas-says,createaseparatefolderforeachassay.
6. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.
Data Analysis
• TheFCSfilegeneratedonaflowcytometershouldbeanalyzedusingBio-Legend’s LEGENDplexTMDataAnalysisSoftware.TheLEGENDplexTM Data AnalysisSoftwarecanbedownloadedforfreehere:www.biolegend.com/legendplex.
• ForPCusers,installthesoftwareonaPCrunningWindows7orWin-dows8anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedinthiskit.Thedonglehasalicensekeystoredinitandisneededtorunthesoftware.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunch-ingthesoftware.
• ForMacusers,installonaMacOSXversion10.7(Lion)orlaterandyouwillbepromotedtorequestasoftwarelicensekeyafterthesoftwareinstallation.
• FollowtheLEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelptousethesoftware(www.bioLegend.com/legendplex; or press F1 for online help at any step of the data analysis).
biolegend.com21
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Chapter 6: ASSAY CHARACTERIZATIONRepresentative Standard Curve
ThisstandardcurvewasgeneratedusingtheLEGENDplexTM Human Acute PhasePanel1fordemonstrationpurposesonly.Astandardcurvemustberunwitheachassay.
5.0
50.0
500.0
5000.0
1 10 100 1000 10000 100000 1000000 10000000
MFI
Concentration (pg/ml)
A4.α2-macroglobulin
A5.α1-AGP
A8. Haptoglobin
A10.α1-antitrypsin
B3.Ceruloplasmin
B4.Fibrinogen
B5.Prothrombin
B7.SAP
Assay SensitivityTheassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.
AnalyteMDC (pg/mL) (n=12)
Mean STDEV
α2-macroglobulin 232.7 89.9
α1-AGP 31.2 12.4
Haptoglobin 6.1 2.7
α1-antitrypsin 26.1 15.4
Ceruloplasmin 107.6 47.2
Fibrinogen 27.7 15.6
Prothrombin 105.4 34.0
SAP 1.4 0.5
Cross-ReactivityTargethumanproteinsweretestedindividuallyattheindicatedconcen-trationsbelowusingtheLEGENDplexTM Human Acute Phase Panel 1,withnegligiblecross-reactivityobservedfornon-intendedtargets.
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
22
Analyte Conc. (ng/mL)
α2-macroglobulin 40,000
α1-AGP 4,000
Haptoglobin 1,000
α1-antitrypsin 4,000
Ceruloplasmin 16,000
Fibrinogen 4,000
Prothrombin 10,000
SAP 200
Thefollowingrecombinantproteinsweretestedindividuallyatatleast50ng/mL.Noornegligiblecross-reactivitywasfound.
Human
PAI-1 SP-D Adiponectin MCP-1
Ferritin BPI Adipsin IL-1β
CRP LBP NGAL IL-6
Properdin CD14 MMP-2 IL-8
tPA Procalcitonin OPN IL-10
SAA MIF MPO IL-12 (p70)
α1-antichymotrypsin CD141 IGFBP-4 IL-17A
β2-microglobulin α1-microglobulin ICAM-1 IL-18
Myoglobin suPAR VCAM-1 IL-23
Antithrombin Leptin MMP-9 IL-33
Plasminogen Proinsulin CystatinC TNF-α
FactorXIII RBP4 Myoglobin IFNγ
PTX3 Resistin MRP8/14 IFNα2
Accuracy (Spike Recovery)
Forspikerecoveryincellculturemedium(n=2),RPMIorDMEMwith10%FBSwerespikedwithtargetrecombinantproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andmea-suredconcentrationswerecomparedwiththeexpectedvalues.
biolegend.com23
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Analyte % of Spike Recovery
α2-macroglobulin 102%
α1-AGP 95%
Haptoglobin 96%
α1-antitrypsin 94%
Ceruloplasmin 92%
Fibrinogen 97%
Prothrombin 95%
SAP 94%
Linearity of Dilution
Serum(n=10)andplasma(n=28)sampleswereinitiallydiluted20,000-foldwithAssayBuffer,thenseriallydiluted2,4,and8foldwithAssayBufferand assayed.
Cellculturesamples(n=2)werespikedwithtargetproteinswithknownconcentrationsintheassayrange,thenseriallydiluted2,4,and8foldwithAssayBufferandassayed.
Themeasuredconcentrationsofseriallydilutedsampleswerethencomparedwiththeconcentrationofthelowestdilutionbasedonserialdilutionfactorused.
Analyte% Linearity
Serum Plasma Cell Culture
α2-macroglobulin 102% 101% 86%
α1-AGP 97% 102% 111%
Haptoglobin 92% 96% 101%
α1-antitrypsin 104% 108% 107%
Ceruloplasmin 112% 93% 128%
Fibrinogen NA* 92% 90%
Prothrombin NA* 82% 92%
SAP 109% 105% 101%*Serum is not claimed as a sample type for Fibrinogen and Prothrombin
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
24
Intra-Assay Precision
Twosampleswithdifferentconcentrationsofeachtargetproteinwereanalyzedinoneassaywith16replicatespersample.Theintra-assayprecisionisshownbelow.
Analyte Sample Mean (pg/mL) STDEV %CV
α2-macroglobulinSample 1 8,744.5 370.8 4%
Sample 2 35,240.5 1,235.5 4%
α1-AGPSample 1 870.4 53.1 6%
Sample 2 3,636.5 129.8 4%
HaptoglobinSample 1 252.0 8.5 3%
Sample 2 946.1 29.2 3%
α1-antitrypsinSample 1 945.2 48.5 5%
Sample 2 3,668.5 137.8 4%
CeruloplasminSample 1 3,598.6 169.9 5%
Sample 2 13,861.8 637.2 5%
FibrinogenSample 1 1,168.4 50.7 4%
Sample 2 4,474.9 188.4 4%
ProthrombinSample 1 2,509.0 144.5 6%
Sample 2 10,141.4 497.5 5%
SAPSample 1 58.7 4.2 7%
Sample 2 224.8 14.3 6%
Inter-Assay Precision
Twosampleswithdifferentconcentrationsofeachtargetproteinwereanalyzedinsixindependentassayswithfourreplicatespersample.Theinter-assayprecisionisshownbelow.
Analyte Sample Mean (pg/mL) STDEV %CV
α2-macroglobulinSample 1 12,363.9 2,196.6 18%
Sample 2 46,666.9 8,082.5 17%
α1-AGPSample 1 1,190.7 221.4 19%
Sample 2 4,676.7 666.0 14%
biolegend.com25
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
HaptoglobinSample 1 318.9 50.8 16%
Sample 2 1,119.4 166.7 15%
α1-antitrypsinSample 1 1,284.2 218.3 17%
Sample 2 4,671.0 722.9 15%
CeruloplasminSample 1 4,816.7 982.5 20%
Sample 2 17,926.3 3,221.1 18%
FibrinogenSample 1 1,525.9 298.5 20%
Sample 2 5,420.4 1,074.9 20%
ProthrombinSample 1 3,783.0 855.6 23%
Sample 2 13,069.8 2,469.0 19%
SAPSample 1 85.7 23.7 28%
Sample 2 291.5 63.8 22%
Biological Samples
Thevaluesinthissectionareprovidedforreferenceonly.Theassayspro-videdinthiskitareintendedforresearchuseonly.
Serum and plasma (samples are paired)
Normalhumanserumsamples(n=20)weretestedforendogenouslevelsoftheproteins.Theconcentrationsareshownbelow.
Analyte Range (ng/mL) % Detectable Mean (ng/mL)
α2-macroglobulin 1029.6-7730.9. 100% 2466.7
α1-AGP 384.3-2489.0 100% 925.9
Haptoglobin 77.3-3061.1 100% 1200.5
α1-antitrypsin 577.5-4823.8 100% 1386.8
Ceruloplasmin 209.0-848.9 100% 416.9
SAP 6.8-40.2 100% 20.3
Serum is not claimed as a sample type for Fibrinogen and Prothrombin
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
26
Normalhumanplasma(Heparin,Citrate,andEDTA)samples(n=60)weretestedforendogenouslevelsoftheproteins.Theconcentrationsareshownbelow.
Analyte Range (μg/mL) % Detectable Mean (μg/mL)
α2-macroglobulin 615.9-4761.8 100% 2100.4
α1-AGP 168.6-2129.4 100% 726.5
Haptoglobin 48.7-3644.1 100% 1175.5
α1-antitrypsin 300.7-3022.8 100% 1088.5
Ceruloplasmin 75.6-1388.1 100% 417.7
Fibrinogen 176.4-7101.6 100% 2431.3
Prothrombin 20.6-256.3 100% 120.7
SAP 2.8-47.2 100% 16.6
Cell Culture Supernatant
Human HepG2 cells (1x106cells/mL)wereculturedunderLPS(1µg/mL),IL-6(10ng/mL),IL-1β(10ng/mL),andTNF-α(25ng/mL)stimulationswithunstimulatedcellsasacontrol.CellCulturesupernatantswerecollected 24hourafterstimulationand assayed. The results (all ng/mL) are sum-marizedbelow.
Analyte Control LPS IL-6 IL-1β TNF-α
α2-macroglobulin 170.0 199.9 266.6 523.3 264.2
α1-AGP 552.3 465.6 620.8 973.4 637.8
Haptoglobin 259.5 224.7 1,286.0 827.9 403.2
α1-antitrypsin 1,298.0 1,165.0 1,286.0 1,346.0 1,241.0
Ceruloplasmin 194.6 167.0 248.1 437.1 273.8
Fibrinogen 330.8 259.5 1,055.0 820.5 271.4
Prothrombin 465.6 371.8 449.1 1,165.0 518.6
SAP 7.3 6.9 7.5 7.8 7.2
biolegend.com27
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
TROUBLESHOOTING
Problem Possible Cause Solution
Bead popula-tionshiftingupwardordownwarddur-ingacquisition
The strong PE signal from high concentra-tionsamplesorstan-dards may spill over to classificationChannel(e.g.,FL3/FL4/APC)and mess up the bead separation.
OptimizeinstrumentsettingsusingKitSetupBeads,andmakeappropriatecom-pensationbetweenchannels.
Filterplatewillnot vacuum orsomewellsclogged
Vacuum pressure is insufficientorvacuummanifold does not seal properly.
Increase vacuum pressure such that 0.2 mLbuffercanbesuctionedin3-5seconds.Cleanthevacuummanifoldandmakesurenodebrisonthemanifold.Pressdowntheplateonthemanifoldtomakeagoodseal.
Samples have insoluble particlesorsampleistooviscous(e.g.,serumand plasma samples)
Centrifugesamplesjustpriortoassaysetup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Ifsomewellsarestillcloggedduringwash-ing,trythefollowing:
1).Addbuffertoallthewells,pipetteupanddownthecloggedwellsandvacuumagain.
2).Useapieceofcleanwipe,wipetheun-dersideofthecloggedwellsandvacuumagain.
3).Takeathinneedle(e.g.,insulinneedle),whileholdingtheplateupward,pokethelittleholeundereachofthecloggedwellsandvacuumagain.Donotpoketoohardortoodeepasitmaydamagethefilterandcauseleaking.
Filterplatewasusedwithoutpre-wet.
Pre-wetplatewithwashbufferbeforerun-ning the assay.
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
28
Insufficientbead count or slowreading
Beads inappropriately prepared
Sonicatebeadvialsandvortexjustpriortoaddition.Agitatemixedbeadsintermit-tentlyinreservoirwhilepipettingthisintothe plate.
Samples cause beads aggregationduetoparticulatematterorviscosity.
Centrifugesamplesjustpriortoassaysetup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Beadswerelostduringwashingforin-tubeassay
Makesurebeadsarespundownbyvisu-allycheckthepellet(beadsareinlightblueorbluecolor).Beverycarefulwhenremovingsupernatantduringwashing.
Probe might be par-tiallyclogged.
Sampleprobemayneedtobecleaned,orifneeded,probeshouldberemovedandsonicated.
Plateleaked
Vacuum pressure set too high
Adjustvacuumpressuresuchthat0.2mLbuffercanbesuctionedin3-5seconds.Donot exceed 10” Hg of vacuum.
Plate set directly on tableorabsorbenttow-elsduringincubationsorreagentadditions
Set plate on plate holder or raised edge sobottomoffilterisnottouchinganysurface.
Liquid present on the under side of the plate aftervacuum
Afterwashing,pressdownplatefirmlyonastackofcleanpapertowelstodrytheunderside of the plate.
Pipettetouchinganddamagedplatefilterduringadditions.
Pipettetothesideofwells.
HighBack-ground
Backgroundwellswerecontaminated
Avoidcross-wellcontaminationbychang-ingtipsbetweenpipettingwhenperform-ingtheassayusingamultichannelpipette.
InsufficientwashesThebackgroundmaybeduetonon-specificbindingofSA-PE.Increasenumberofwashes.
Debris (FSC/SSC) during sample acquisi-tion
Debris or platelet may exist in sample solu-tion.
Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.
biolegend.com29
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Variationbe-tweenduplicate samples
Beadsaggregation Sonicate and vortex the Beads prior to use.
Multichannelpipettemay not be calibrated or inconsistent pipet-ting
CalibratePipette.Ensuregoodpipettingpractice.Primepipettebeforeusemayhelp.
Platewashingwasnotuniform
Makesureallreagentsarevacuumedoutcompletelyinallwashsteps.
Samples may contain particulatematters.
Centrifugesamplesjustpriortoassaysetup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Loworpoorstandard curve signal
Thestandardwasin-correctlyreconstituted,stored or diluted
Followtheprotocoltoreconstitute,storeanddilutestandard.Doublecheckyourcalculation.
Wrongorshortincuba-tiontime
Ensurethetimeofallincubationswasappropriate.
Signals too high,standardcurves satu-rated
PMT value for FL2/PE set too high
MakesurethePMTsettingforthere-porter channel is appropriate
Plateincubationtimewastoolong Useshorterincubationtime.
Sample read-ings are out of range
Samples contain no or belowdetectablelevelsof analyte
Makesuretheexperimenttogeneratethesamplesworked.Useproperpositivecontrols.
Samplesconcentrationshigher than highest standard point.
Dilutesamplesandanalyzeagain.
Standardcurvewassaturated at higher end of curve.
MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoolong
Missed beads populationsduringreading,ordistributionis unequal
Sample may cause some beads to ag-gregate.
Centrifugesamplesjustpriortoassaysetup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Beadspopulationsarenot mixed properly
Makesureallbeadpopulationsaremixed.and in similar numbers.
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
30
Notes
biolegend.com31
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
Tel: 858-768-5800
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
32
Notes
biolegend.com33
LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel
PLA
TE M
AP
(for
in-p
late
ass
ay)
1
2 3
4 5
6 7
8 9
10
11
12
A
C0
C4
Sa
mpl
e1
Sa
mpl
e5
Sa
mpl
e9
Sa
mpl
e 13
Sa
mpl
e17
Sa
mpl
e21
Sa
mpl
e25
Sa
mpl
e29
Sa
mpl
e33
Sa
mpl
e 37
B
C0
C4
Sa
mpl
e1
Sa
mpl
e5
Sa
mpl
e9
Sa
mpl
e 13
Sa
mpl
e17
Sa
mpl
e21
Sa
mpl
e25
Sa
mpl
e29
Sa
mpl
e33
Sa
mpl
e37
C
C1
C5
Sa
mpl
e2
Sa
mpl
e6
Sa
mpl
e10
Sa
mpl
e 14
Sa
mpl
e18
Sa
mpl
e22
Sa
mpl
e26
Sa
mpl
e30
Sa
mpl
e34
Sa
mpl
e38
D
C1
C5
Sa
mpl
e2
Sa
mpl
e6
Sa
mpl
e10
Sa
mpl
e 14
Sa
mpl
e18
Sa
mpl
e22
Sa
mpl
e26
Sa
mpl
e30
Sa
mpl
e34
Sa
mpl
e38
E
C2
C6
Sa
mpl
e3
Sa
mpl
e7
Sa
mpl
e11
Sa
mpl
e 15
Sa
mpl
e19
Sa
mpl
e23
Sa
mpl
e27
Sa
mpl
e31
Sa
mpl
e35
Sa
mpl
e39
F
C2
C6
Sa
mpl
e3
Sa
mpl
e7
Sa
mpl
e11
Sa
mpl
e 15
Sa
mpl
e19
Sa
mpl
e23
Sa
mpl
e27
Sa
mpl
e31
Sa
mpl
e35
Sa
mpl
e39
G
C3
C7
Sa
mpl
e4
Sa
mpl
e8
Sa
mpl
e12
Sa
mpl
e 16
Sa
mpl
e20
Sa
mpl
e24
Sa
mpl
e28
Sa
mpl
e32
Sa
mpl
e36
Sa
mpl
e40
H
C3
C7
Sa
mpl
e4
Sa
mpl
e8
Sa
mpl
e12
Sa
mpl
e 16
Sa
mpl
e20
Sa
mpl
e24
Sa
mpl
e28
Sa
mpl
e32
Sa
mpl
e36
Sa
mpl
e40
LEGENDplex™ Kits are manufactured by BioLegend 8999 BioLegend WaySan Diego, CA 92121Tel: 1.858.768.5800Tel: US & Canada Toll-Free: 1.877.Bio-Legend (1.877.246.5343)Fax: 1.877.455.9587Email: [email protected]
For a complete list of world-wide BioLegend offices and distributors, please visit our website at: biolegend.com
Enabling Legendary Discovery™
750000670_V01