LEGENDplex™...A4 A6 A10 B4 B5 B6 B7 B3 B9 B2 Tel: 858-768-5800 LEGENDplexTM Human Immunoglobulin Isotyping Mix and Match Subpanel 6 Storage Information Recommended storage for all

LEGENDplex™Mul�-Analyte Flow Assay Kit

Enabling Legendary Discovery™

For Accurate Quantification of Multiple Human Th(T helper Cell) Cytokines from Cell Culture Supernatant,

Serum, Plasma and Other Biological Samples

Please read the entire manual before running the assay

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LEGENDplex™Mul�-Analyte Flow Assay Kit


Human Immunoglobulin Isotyping 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.

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LEGENDplexTM Human Immunoglobulin Isotyping 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……………………………………………………..........

LinearityofDilution………………………………………………..........

Intra-AssayPrecision……………………………………...................

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Inter-AssayPrecision……………………………………...................

BiologicalSamples…………………………………………….………....

TROUBLESHOOTING.........……………………………………………………....

PLATE MAP...................................................................................

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Chapter 1: KIT DESCRIPTIONIntroductionImmunoglobulins (Igs) play important roles in immune responses by recogniz-ing,bindingandneutralizingspecificantigenssuchasbacteria,virusesandtox-ins.Inaddition,immunoglobulinscanbindandmarkthepathogens,aprocesscalledopsonizationthroughwhichphagocytescanthenengulfandeliminatethepathogens.Duringimmuneresponses,plasmacellscanswitchfromproduc-ing one immunoglobulin class to another by changing the amino acid sequence intheconstantregionoftheheavychain.Therearefivedifferentclassesofimmunoglobulins: IgM, IgG, IgE, IgA and IgD. IgG and IgA can be further divided intodifferentsubclassesbasedonthedifferenceinthenumberofdisulfidebondsandthelengthandflexibilityofthehingeregion.Perturbationstotheexpression levels of the immunoglobulin classes and subclasses are associated withdifferentdiseasessuchaslympho-proliferationorimmunoglobulin-defi-ciencydisorders.Moreover,quantitationoftheseclassesandsubclassesduringandaftervaccinationcouldprovideveryusefulinformationaboutprimaryandsecondary immune responses to a vaccine.

The LEGENDplexTM Human Immunoglobulin Isotyping Panel is a bead-based multiplexassaypanel,usingfluorescence-encodedbeadssuitableforuseonvariousflowcytometers.Itallowsforsimultaneousquantificationof8humanimmunoglobulins, including IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM. This assaypanelprovideshigherdetectionsensitivityandbroaderdynamicrangethantraditionalELISAmethods.

The LEGENDplexTMHumanImmunoglobulinIsotypingPanelsareconfiguredasshownbelow:

Catalog No.

Plex Size Targets Recommended

Sample TypeRecommended Dilution Factor

740637 740638 8-plex IgG1, IgG2, IgG3, IgE,

IgG4, IgA, IgM, IgD Tissue culture Varies

740639740640 6-plex IgG1, IgG2, IgG3, IgG4,

IgA, IgM Serum, Plasma 100,000

740714740715 4-plex IgG1, IgG2, IgG3, IgG4 Serum, Plasma 100,000

740641740642 1-plex IgE Serum, Plasma 100

740643740644 1-plex IgD Serum, Plasma 5,000

The LEGENDplexTMHumanImmunoglobulinIsotypingPanelisdesignedtoallowflexiblecustomizationwithinthepanel.Pleasevisitwww.biolegend.com/leg-endplexformoreinformationonhowtomixandmatchwithinthepanel.This assay is for research use only.

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Principle of the Assay

BioLegend’s LEGENDplexTM assays are bead-based immunoassays using the samebasicprincipleassandwichimmunoassays.

Beadsaredifferentiatedbysizeandinternalfluorescenceintensities.Eachbeadsetisconjugatedwithaspecificantibodyonitssurfaceandservesasthecap-turebeadsforthatparticularanalyte.Whenaselectedpanelofcapturebeadsismixedandincubatedwithasamplecontainingtargetanalytesspecifictothecaptureantibodies,eachanalytewillbindtoitsspecificcapturebeads.Afterwashing,abiotinylateddetectionantibodycocktailisadded,andeachdetec-tionantibodyinthecocktailwillbindtoitsspecificanalyteboundonthecap-turebeads,thusformingcapturebead-analyte-detectionantibodysandwiches.Streptavidin-phycoerythrin(SA-PE)issubsequentlyadded,whichwillbindtothebiotinylateddetectionantibodies,providingfluorescentsignalintensitiesinproportiontotheamountofboundanalytes.

Sincethebeadsaredifferentiatedbysizeandinternalfluorescenceintensityonaflowcytometer,analyte-specificpopulationscanbesegregatedandPEfluorescentsignalquantified.Theconcentrationofaparticularanalyteisdeter-mined using a standard curve generated in the same assay.

Beads Usage

The LEGENDplexTM bead-based assay systemusestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.Theinternaldye can be detected using FL3, FL4, or APC channel, depending on the type of flowcytometerused.ThesmallerBeadsAconsistsof6beadpopulationsandthelargerBeadsBconsistsof7beadpopulations(Figure2-3).

Using8outofthe13beadpopulationsdistinguishedbysizeandinternalfluo-rescent dye, the Human Immunoglobulin IsotypingPanelallowssimultaneousdetectionof8immunoglobulinsinasinglesample.Eachanalyteisassociatedwithaparticularbeadsetasindicated(Figures2-3andTable1). Figure 1. Beads Differentiated by Size

Beads A = smaller beads

Beads B = larger beads

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Figure 2. Beads A Classification by FL4

Figure 3. Beads B Classification by FL4

ForBeadsusageinthepanel,pleaserefertoTable1below:

Table 1. Panel Targets and Bead ID

Target Bead IDIsotyping (8-plex)Cat No.

740637 or 740638

Isotyping (6-plex)Cat. No.

740639 or 740640

Isotyping IgGs (4-plex)

Cat. No. 740639 or

740640

Isotyping IgE (1-plex)

Cat. No. 740641 or

740642

Isotyping IgD (1-plex)

Cat. No. 740643 or

740644

Top Standard Concentrations

IgG1 A4 √ √ √ The top standard concentrationof each target may vary and

may subject to change from lot

to lot. Please refer to the lot-specificCertificateof

Analysis for this information

IgG2 A5 √ √ √

IgG3 A6 √ √ √

IgE A7 √ √

IgG4 B3 √ √ √

IgA B4 √ √

IgM B5 √ √

IgD B6 √ √

*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, A6...B2, B3, B4...). Please refer to the LEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelp for details (www.biolegend.com/legendplex).

A5 A7 A8

A4

A6

A10

B4 B5

B6 B7

B3

B9

B2

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Storage Information

Recommendedstorageforalloriginalkitcomponentsisbetween2°Cand8°C.DONOTFREEZEBeads,DetectionAntibodiesorSA-PE.

• Oncethestandardshavebeenreconstituted,immediatelytransfercon-tentsintopolypropylenevials.DONOTSTORERECONSTITUTEDSTAN-DARDS IN GLASS VIALS.

• Uponreconstitution,leftoverstandardshouldbestoredat≤-70°Cforusewithinonemonth.Avoidmultiple(>2)freeze-thawcycles.Discardanyleftoverdilutedstandards.

Materials Supplied

The LEGENDplexTMkitcontainsreagentsfor100tests,listedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.

For the Mix and Match Subpanels, individual beads are provided at 13X con-centration.TheBufferSetcontainsSetupBeads,allBuffers,PlateSealers,SA-PEandDataAnalysisSoftwareDongle.AmanualisalsoprovidedforeachMixandMatch subpanel.

Kit Components Quantity Volume Cat. #

Capture Beads* (see tables below for more information) varies varies varies

LEGENDplex™ Human Immunoglobulin IsotypingPanelDetectionAntibodies 1bottle 3.5 mL 740645

LEGENDplex™ Human Immunoglobulin Isotyping Panel Standard 1 vial lyophi-

lized 740646

LEGENDplex™BufferSetK 1 740713

Filter Plate** or V-bottomPlate*** 1 Plate 740377**or

740379***

Human Immunoglobulin Isotyping Mix and Match Subpanel Manual 1 75464

**Forassaywithfilterplate.***ForassaywithV-bottomplate.

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Capture beads for Mix and Match Subpanels*:

Bead Name Quantity Volume Cat. #LEGENDplex™ Human IgG1 Capture Bead A4, 13X 1 vial 270 µL 740647

LEGENDplex™ Human IgG2 Capture Bead A5, 13X 1 vial 270 µL 740648

LEGENDplex™ Human IgG3 Capture Bead A6, 13X 1 vial 270 µL 740649

LEGENDplex™ Human IgE Capture Bead A7, 13X 1 vial 270 µL 740650

LEGENDplex™ Human IgG4 Capture Bead B3, 13X 1 vial 270 µL 740651

LEGENDplex™ Human IgA Capture Bead B4, 13X 1 vial 270 µL 740652

LEGENDplex™ Human IgM Capture Bead B5, 13X 1 vial 270 µL 740653

LEGENDplex™ Human IgD Capture Bead B6, 13X 1 vial 270 µL 740654

* Please refer to Beads ID and Panel-Specific Target Selection table (Table 1), to see which capture beads are included in each panel.

LEGENDplex™ Buffer Set K (Cat#: 740713)

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 3bottle 25 mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564DataAnalysisSoftwareDongle 1 21217Plate Sealers 4 sheets 78101

No plate is included in Buffer Set K. Plate need to be ordered separately. Please order the correct type of plate based on the preferred assay protocol (Cat# 740377 or 740378 for Filter Plate and Cat# 740379 for V-bottom Plate)

Materials to be Provided by the End-User

• Aflowcytometerequippedwithtwolasers(e.g.,a488nmbluelaseror532nmgreenlaseranda633-635nmredlaser)capableofdistinguishing575 nm and 660 nm oraflowcytometerequippedwithonelaser(e.g.,488nmbluelaser)capableofdistinguishing575nmand670nm.

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Partial list of compatible flow cytometers:

Flow Cytometer

Reporter Channel

ChannelEmission

Classification Channel

Channel Emission

Compensa-tion needed?

BD FACSCaliburTM

(single laser)FL2 575 nm FL3 670 nm Yes

BD FACSCaliburTM

(dual laser)FL2 575 nm FL4 660 nm No*

BD FACSArrayTM Yellow 575 nm Red 660 nm No*

BD AccuriTM C6 FL2 585 nm FL4 675 nm No*

BD FACSCantoTM

BD FACSCantoTM IIPE 575 nm APC 660 nm No*

BDTM LSR, LSR IIBD LSRFortessaTM PE 575-585

nm APC 660 nm No*

BD FACSAriaTM PE 575 nm APC 660 nm No*

Beckman Coulter-CytoFLEX PE 585 nm APC 660 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)

• Laboratory vortex mixer

• Sonicator bath (e.g., Branson Ultrasonic Cleaner model #B200, or equiva-lent)

• Aluminum foil

• Absorbentpadsorpapertowels

• Plateshaker(e.g.,Lab-LineInstrumentsmodel#4625,orequivalent)

• Tabletop centrifuges (e.g., Eppendorf centrifuge 5415 C, or equivalent)

If the assay is performed in a filter plate (recommended);

• Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,cat#MSVMHTS00orequivalent).Instructionsonhowtousethevacuummanifoldcanbefoundatthesupplier’swebsite.

• A vacuum source (mini vacuum pump or line vacuum, e.g., Millipore

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VacuumPump,catalog#WP6111560,orequivalent)

• Ifneeded,additionalFilterplatecanbeorderedfromBioLegend(Cat#740377 or 740378)

If the assay is performed in a V-bottom plate (optional);

• Centrifugewithaswingingbucketadaptorformicrotiterplates(e.g.,Beck-man Coulter AllegraTM6RCentrifugewithMICROPLUSCARRIERadaptorforGH3.8 and JS4.3 Rotors) .

• Ifneeded,additionalV-bottomplatecanbeorderedfromBioLegend(Cat#740379)

Precautions

• All blood components and biological materials should be handled as poten-tiallyhazardous.FollowuniversalprecautionsasestablishedbytheCenterforDiseaseControlandPreventionandbytheOccupationalSafetyandHealthAdministrationwhenhandlinganddisposingofinfectiousagents.

• Sodiumazidehasbeenaddedtosomereagentsasapreservative.Al-thoughtheconcentrationsarelow,sodiumazidemayreactwithleadandcopperplumbingtoformhighlyexplosivemetalazides.Ondisposal,flushwithalargevolumeofwatertopreventazidebuild-up.

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandBeadsarelight-sensitive.Minimizelightexposure.

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Chapter 2: ASSAY PREPARATION

Sample Collection and HandlingPreparation of Serum Samples:

• Allowthebloodtoclotforatleast30minutesandcentrifugefor20min-utes at 1,000 x g.

• Remove serum and assay immediately or aliquot and store samples at ≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.

Preparation of Plasma Samples:

• Plasmacollectionusingananti-coagulant(e.g.EDTA,Heparin,Citrate)isrecommended. Centrifuge for 20 minutes at 1,000 x gwithin30minutesofbloodcollection.

• Remove plasma and assay immediately, or aliquot and store samples at ≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedwellandcentrifugedtoremoveparticulates.

Preparation of Tissue Culture Supernatant:

• Centrifuge the sample to remove debris and assay immediately. If not pos-sible,aliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

Reagents PreparationPreparation of Antibody-Immobilized Beads

Theindividualbeads(13X)shouldbemixedwitheachotheranddilutedto1XfinalconcentrationwithAssayBufferpriortouse.Tomixthebeads,followthestepsbelow(a5-plexsubpanelisusedasanexample):

1. Sonicate the beads vials for 1 minute in a sonicator bath and then vortex for 30 seconds to completely resuspend the beads.

2. Calculate the amount of mixed and diluted beads needed for the assay.Prepareextratocompensateforpipettingloss.Eachreactionneeds25µLofmixedanddilutedbeads.For50reactions,prepare1.5mLofmixedbeads.For96reactions,prepare3mLofmixedbeads.

3. Tomake1.5mlof5-plex1Xdilutedbeads,transfer115µLofeachofthe 5 individual beads (13X) to a fresh tube (total bead volume = 575 µL)andadd925µLofAssayBuffertomakethefinalvolumeof1.5mL.

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Preparation of Wash Buffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

Standard Preparation

1. Priortouse,reconstitutethelyophilizedHumanImmunoglobulinIsotyp-ingPanelStandardCocktail,with250µLAssayBuffer.

2. Mixandallowthevialtositatroomtemperaturefor15minutes,andthen transfer the standard to an appropriately labeled polypropylene microfugetube.ThiswillbeusedasthetopstandardC7.

Note: The top standard concentrations of analytes in this panel was set at various concentrations, but may be subject to change from lot to lot (see lot-specific Certificate of Analysis provided with kit box for details).

3. Label 6 polypropylene microfuge tubes as C6, C5, C4, C3, C2 and C1, respectively.

4. Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthe top standard by transferring 25 µL of the top standard C7 to the C6 tubeandmixwell.ThiswillbetheC6standard.

Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2and C1 standards (see the table below using the top standard at 400,000 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 -- -- -- 400,000

C6 1:4 75 25 µL of C7 100,000

C5 1:16 75 25 µL of C6 25,000

C4 1:64 75 25 µL of C5 6250

C3 1:256 75 25 µL of C4 1562.5

C2 1:1024 75 25 µL of C3 390.6

C1 1:4096 75 25 µL of C2 97.6

C0 -- 75 -- 0

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Sample DilutionFor cell culture supernatant samples, the levels of analyte can vary greatly fromsampletosample.Whilethesamplescanbetestedwithoutdilutions,a preliminary experiment may be required to determine the appropriate dilutionfactor.Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasurement.

Forserumandplasmasamples,followtargetspecificdilutionrecommen-dationsbelow.

Target Recommended Sample Type

Recommended Dilution Factor

IgG1 Serum, Plasma 100,000



IgE Serum, Plasma 100


IgA Serum, Plasma 100,000

IgM Serum, Plasma 100,000

IgD Serum, Plasma 5,000

• Recommended dilution scheme;

Sample 1st Dilution(1:200)

2nd Dilution(1:500)

Final dilution fold

Serum, plasma

2 µL + 398 µL AssayBuffer

2 µL1stDilution+998µLBuffer

100,000

Sample 1st Dilution(1:100)

2nd Dilution(1:50)

Final dilution fold

Serum, plasma

2 µL + 198 µL AssayBuffer

4 µL1stDilution+196µLAssayBuffer

5,000

Sample Dilution(1:100)

Final dilution fold

Serum, plasma

2 µL+198µLAssayBuffer 100

Iffurthersampledilutionisdesired,dilutionshouldbedonewithAssayBuf-fer to ensure accurate measurement.

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Chapter 3: ASSAY PROCEDURE

The LEGENDplexTM assaycanbeperformedinafilterplate,orinaV-bottomplate.

• Thein-filterplateassayprocedureisrecommendedduetoitsgoodsample to sample consistency, assay robustness and ease of handling. This procedurerequiresavacuumfiltrationunitforwashing(seeMaterials to be Provided by the End-User, page 7). If you have performed bead-based multiplexassaysbefore,yourlabmayalreadyhavethevacuumfiltrationunit set up.

• Ifthein-filterplateassayprocedureisnotpossibleorifyouprefer,theas-saycanbeperformedinaV-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 29). 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.Toremovethe excess volume, place the plate on the vacuum manifold and apply vacuum.Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypressingtheplateonastackofcleanpapertowels.Placetheplateontopof the inverted plate cover.

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Loadtheplateasshowninthetablebelow(intheorderfromlefttoright):AssayBuffer Standard Sample*

StandardWells 25 µL 25 µL ---

Samplewells 25 µL --- 25 µL *See Sample Dilution

2. Vortexmixedbeadsbottlefor30seconds.Add25μLofmixedbeadstoeachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringadditionofthebeads,shakemixedbeadsbottleintermit-tentlytoavoidbeadsettling).

3. Sealtheplatewithaplatesealer.To avoid plate leaking, do not apply posi-tive pressure to the sealer when sealing the plate.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshaker,secureitandshakeatapproximate500rpmfor2hoursatroom temperature.

4. Do not invert the plate! Place the plate on the vacuum manifold and apply vacuumasbeforeinStep1.Add200µLof1XWashBuffertoeachwell.RemoveWashBufferbyvacuumfiltration.BlotexcessWashBufferfromthebottomoftheplatewithanabsorbentpadorpapertowels. Repeat this washingsteponcemore.

5. Add25µLofDetectionAntibodiestoeachwell.

6. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximately500rpmfor1houratroomtemperature.

7. Do not vacuum!Add25µLofSA-PEtoeachwelldirectly.

8. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximate500rpmfor30minutesatroomtemperature.

9. Repeat step 4 above.

10. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplateshakerfor1minute.

11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note: Prolonged sample storage can lead to reduced signal).

Iftheflowcytometerisequippedwithanautosampler,readtheplatedirectly using the autosampler. Please be sure to program the autosampler to resuspend beads in the well immediately before taking samples. The

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probe height may need to be adjusted when using an autosampler.

If an autosampler is not available, the samples can be transferred from the filterplateto micro FACS (or FACS) tubes and read manually.

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

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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,page29).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,theori-entationandreadingsequenceshouldbecarefullyplanned.

1. Loadtheplateasshowninthetablebelow(intheorderfromlefttoright):AssayBuffer Standard Sample*

StandardWells 25 µL 25 µL ---

Samplewells 25 µL --- 25 µL

*See Sample Dilution

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 so it causes 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 7). Donotuseexcessivecentrifugationspeedasitmaymakeithardertoresuspendbeadsinlatersteps.Make sure the timer of the centrifuge works properly and standby to make sure the centrifuge reaches preset speed.

5. Immediatelyaftercentrifugation,dumpthesupernatantintoasinkbyquicklyinvertingandflickingtheplatein one continuous and forceful mo-tion.Donotworryaboutlosingbeadsevenifthepelletisnotvisible.Thebeadswillstayinthetipofthewellnicely.

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6. Blottheplateonastackofcleanpapertowelanddraintheremainingliquidfromthewellasmuchaspossible.Becarefulnottodisturbthebeadpellet.

Alternatively,removalofthesupernatantmaybecompletedusingamultichannelpipettesetat75µL. Try to remove as much liquid as possible withoutremovinganybeads. Be sure to changepipettetipsbetweeneachroworcolumn.

7. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Asecondwashisoptional,butmayhelpreducebackground.

8. Add25µLofDetectionAntibodiestoeachwell.

9. Sealtheplatewithanewplatesealer.Covertheentireplatewithalumi-num foil to protect the plate from light. Shakeat800rpmonaplateshakerfor 1 hour at room temperature.

10. Do not wash the plate!Add25µLofSA-PEtoeachwelldirectly.

11. Sealtheplatewithanewplatesealer.Wraptheentireplatewithaluminumfoilandshaketheplateonaplateshakeratapproximate800rpmfor30minutes at room temperature.

12. Repeat step 4 and 5.

13. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Thiswashingstepisoptionalbutithelpstoreducethebackground.

14. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsbypipet-ting.

15. 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.

If an autosampler is not available, the samples can be transferred from the plate to micro FACS (or FACS) tubes and read manually.

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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

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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.Setthenumberofbeadstobeacquiredtoabout300 per analyte (e.g., acquire 2,400 beads for a 8-plex assay or 4000 beads for a 13-plex assay). Do not set to acquire total events as samples may contain large amounts of debris. Instead, create a large gate to include both Beads 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,readsamplesinthesameorderasshownonthePLATEMAPattachedatthe end of the manual. For an in-plate assay, read column by column (A1, B1, C1...A2, B2, C2...).

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Whennamingdatafiles,trytousesimplenameswithaconsecutivenum-bering for easy data analysis (e.g. for standards, C0.001, C0.002, C1.003, C1.004, C2.005, C2.006, C3.007, C3.008, ... C7.015, C7.016; for samples, S1.017,S1.018,S2.019,S2.020,S3.021,S3.022…)

StoreallFCSfilesinthesamefolderforeachassay.Ifrunningmultipleas-says, create a separate folder for each assay.

6. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

Data Analysis

• TheFCSfilegeneratedonaflowcytometershouldbeanalyzedusingBioLegend’s LEGENDplexTMDataAnalysisSoftware.TheLEGENDplexTM Data AnalysisSoftwarecanbedownloadedforfreehere:www.biolegend.com/legendplex.

• ForPCusers,installthesoftwareonaPCrunningWindows7orWindows8anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedinthiskit.Thedonglehasalicensekeystoredinitandisneededtorunthesoftware.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunchingthesoftware.

• ForMacusers,installonaMacrunningMacOSXversion10.7(Lion)orlaterandyouwillbepromotedtorequestasoftwarelicensekeyafterthesoftwareinstallation.

• FollowtheLEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelptousethesoftware(www.bioLegend.com/legendplex; or press F1 for online help at any step of the data analysis).

biolegend.com21


Chapter 6: ASSAY CHARACTERIZATION

Representative Standard Curve ThisstandardcurvewasgeneratedusingtheLEGENDplexTM Human Immu-

noglobulinIsotypingPanelfordemonstrationpurposesonly.Astandardcurvemustberunwitheachassay.

Assay SensitivityTheminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTMDataAnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.Assaysensitivitypresentedhere is MDC + 2 STDEV.

AnalyteSensitivity

(pg/mL)(N=6)

Human IgG1 21.36 + 39.70

Human IgG2 468.22 + 33.65

Human IgG3 2.28 + 2.97

Human IgE 19.03 + 51.52

Human IgG4 6.32 + 1.47

Human IgA 33.10 + 28.82

Human IgM 177.33 + 36.53

Human IgD 53.93 + 4.89

1

10

100

1000

10000

1 10 100 1000 10000 100000 1000000 10000000

MFI

Concentration (pg/mL)

A4.IgG1

A5.IgG2

A6.IgG3

A7.IgE

B3.IgG4

B4.IgA

B5.IgM

B6.IgD

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22

Cross-ReactivityCrossreactivitywastestedinthepanelusingtheLEGENDplexTM Human ImmunoglobulinIsotypingPanel.Thefollowinghumanimmunoglobulinsweretestedattherespectiveconcentrationasshowninthetablebelow.IgG3crossesupto3%withIgG1assay.Noornegligiblecross-reactivitywasfoundforalltheothertestedanalytes.

Human

Target IgG1 IgG2 IgG3 IgE IgG4 IgA IgM IgD

Concentration (ng/mL)

400 3200 100 800 50 400 1600 400

Mouse

Target IgG1 IgG2a IgG2b IgG3 IgA IgM IgE

Concentration (ng/mL)

500 500 500 500 500 500 500

Linearity of Dilution

Fortestinglinearityofdilution,serumandplasmasampleswerefirstdilutedtotheirrespectivedilutionfactorsasmentionedinSampleDilu-tion,thenseriallydiluted1:2,1:4,1:8withAssayBufferandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerethencomparedwiththatoftheinitiallydilutedsamples.

Analyte Linearity of Dilution (Serum)

Linearity of Dilution (Plasma)

Human IgG1 101% 99%Human IgG2 66% 69%Human IgG3 89% 91%

Human IgE 95% 104%Human IgG4 102% 96%Human IgA 95% 96%Human IgM 98% 98%Human IgD 135% 108%

biolegend.com23


Intra-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinoneassaywith16replicatesforeachsample.Theintra-assayprecisionwascalculatedasbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

Human IgG1Sample 1 2020.94 71.32 4%Sample 2 7792.56 162.44 2%



Human IgESample 1 4190.66 100.12 2%Sample 2 15597.76 285.13 2%


Human IgASample 1 1797.31 62.38 3%Sample 2 6983.05 172.75 2%

Human IgMSample 1 7089.48 220.19 3%Sample 2 26832.52 600.57 2%

Human IgDSample 1 1964.74 44.88 2%Sample 2 7456.15 210.68 3%

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24

Inter-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinfourindependentassayswith4replicatesforeachsample.Theinter-assayprecisionwascalculatedasbelow.

Analyte Sample Mean (pg/mL) STDEV %CV




Human IgESample 1 4673.86 578.75 12%Sample 2 15875.11 1572.28 10%


Human IgASample 1 2152.27 151.25 7%Sample 2 6888.56 747.07 11%

Human IgMSample 1 8269.99 694.93 8%Sample 2 27319.01 2690.52 10%

Human IgDSample 1 2149.19 225.79 11%Sample 2 7260.50 763.26 11%

Biological Samples

Serum and Plasma (Samples are paired)

Normalhumanserumsamples(n=20)weretestedforendogenouslevelsoftheimmunoglobulinisotypes.Theconcentrationsmeasuredareshownbelow.

biolegend.com25


AnalyteRange

(µg/mL)% of

DetectableMedian (µg/mL)

Human IgG1 3239.07 - 20869.79 100% 8006.32

Human IgG2 272.80 - 2086.20 100% 873.30

Human IgG3 140.60 - 1218.38 100% 595.91

Human IgE 0.27 - 7.67 100% 0.73

Human IgG4 45.17 - 3774.34 100% 366.14

Human IgA 1836.76 - 12384.43 100% 4361.83

Human IgM 449.76 - 4175.35 100% 1293.44

Human IgD 0.64 - 37.08 100% 13.59

Normalhumanplasmasamples(n=20)weretestedforendogenouslevelsoftheimmunoglobulinisotypes.Theconcentrationsmeasuredareshownbelow.

AnalyteRange

(µg/mL)% of

DetectableMedian (µg/mL)

Human IgG1 2097.85 - 18171.98 100% 4862.87

Human IgG2 122.26 - 1905.86 100% 579.21

Human IgG3 61.62 - 1281.92 100% 504.39

Human IgE 0.09 - 5.71 100% 0.73

Human IgG4 3.83 - 2805.58 100% 233.07

Human IgA 914.15 - 8485.23 100% 3268.81

Human IgM 313.30 - 2250.03 100% 1088.25

Human IgD 0.57 - 55.41 100% 12.37

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26

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 particlesorsampleistoo viscous (e.g., serum and plasma samples)

Centrifuge samples just prior to assay setup 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.

biolegend.com27


Insufficientbead count or slowreading

Beads inappropriately prepared

Sonicate bead vials and vortex just prior toaddition.Agitatemixedbeadsintermit-tentlyinreservoirwhilepipettingthisintothe plate.

Samples cause beads aggregationduetoparticulatematterorviscosity.


Beadswerelostduringwashingforin-tubeassay

Makesurebeadsarespundownbyvisu-allycheckthepellet(beadsareinlightblueorbluecolor).Beverycarefulwhenremovingsupernatantduringwashing.

Probe might be par-tiallyclogged.

Sample probe may need to be cleaned, or if needed, probe should be removed and sonicated.

Plateleaked

Vacuum pressure set too high

Adjust vacuum pressure such that 0.2 mL buffercanbesuctionedin3-5seconds.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.

Centrifuge samples before analyzing samples. Remove platelet as much as possible.

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28

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.


Loworpoorstandard curve signal

Thestandardwasin-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeanddilutestandard.Doublecheckyourcalculation.

Wrongorshortincuba-tiontime

Ensurethetimeofallincubationswasappropriate.

Signals too high, standard curves satu-rated

PMTvalueforFL2/PEset 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.

Dilute samples and analyze again.

Standardcurvewassaturated at higher end of curve.

MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoolong

Missed beads populationsduring reading, ordistributionis unequal

Sample may cause some beads to ag-gregate.


Beadspopulationsarenot mixed properly

Makesureallbeadpopulationsaremixed.and in similar numbers.

biolegend.com29


PLA

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LEGENDplex™ Kits are manufactured by BioLegend 9727 Pacific Heights Blvd.San 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]

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LEGENDplex™...A4 A6 A10 B4 B5 B6 B7 B3 B9 B2 Tel: 858-768-5800 LEGENDplexTM Human Immunoglobulin Isotyping Mix and Match Subpanel 6 Storage Information Recommended storage for all