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BD Cytometric Bead ArrayMultiplexed Bead-BasedImmunoassays
Cover Illustration: Fairman Studios, LLC.
BD Cytometric Bead Array:Multiplexed Bead-Based ImmunoassaysBD Cytometric Bead Array (CBA) is a flow cytometry application thatallows users to quantify multiple proteins simultaneously. The BD CBAsystem uses the broad dynamic range of fluorescence detection offeredby flow cytometry and antibody-coated beads to efficiently captureanalytes. Each bead in the array has a unique fluorescence intensity sothat beads can be mixed and run simultaneously in a single tube. Thismethod significantly reduces sample requirements and time to resultsin comparison with traditional ELISA and Western blot techniques.BD CBA solutions are designed formultiplexed analysis, to provide moredata using a single sample. Multiplexing isespecially useful when only a small amountof sample is available, maximizing thenumber of proteins that can be analyzed.With BD CBA, up to 30 proteins can beanalyzed using just 25 to 50 μL of sample.Other methods such as ELISA and Westernblot require a similar amount of sample,but only one protein can be analyzedfrom the same volume. With the BD CBAEnhanced Sensitivity Flex Set system, it ispossible to detect as low as 0.274 pg/mL in amultiplexed assay.The BD CBA portfolio includes assaysfor measurement of a variety of solubleand intracellular proteins, includingcytokines, chemokines, growth factors, andphosphorylated cell signaling proteins.BD CBA solutions are available in two formatsto meet diverse needs:BD CBA Flex Sets provide an open andconfigurable method of detection, so thatresearchers can build their own multiplexes.BD CBA Kits are preconfigured for achievingconsistent results for routine panels.Available for most BD flow cytometers,BD CBA solutions combine our leadershipin instrumentation with innovation inapplication development to deliver a flexibleand robust assay system to fulfill diverseresearch requirements.BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.3
BD CBA Flex Sets—Open and ConfigurableSoluble protein assaysBD CBA Soluble Protein Flex Set assays are available for thedetection of cytokines, chemokines, and growth factorsfrom serum, plasma, or tissue culture supernatant samples.These include T-cell differentiation factors, modulators ofinflammation, and other key markers for monitoring theimmune response.The BD CBA Flex Set system provides an open andconfigurable menu of bead-based reagents designed tomake it easy to create multiplex assays.Available specificities include soluble protein assays fordetection of human, mouse, or rat cytokines, chemokines,and growth factors; human immunoglobulins; and cellsignaling assays for detection of phosphorylated cellsignaling proteins.The assays have been formulated to be mixed into any sizeplex, and they are all sold individually, to provide a highlyflexible system. All products are available off-the-shelf socustom orders are not required. Each product area(ie, Human Soluble Protein, Mouse/Rat Soluble Protein,and Human Immunoglobulin) has a unique Master BufferKit. All assays within each product area have been verifiedfor performance in single-plex and in multiplexed scenariosto ensure consistent data.Up to 30 analytes can be measured simultaneously usingthe BD CBA Flex Set system on a flow cytometer equippedwith 488-nm or 532-nm and 633-nm lasers.BeadsSampleConcentrationAnalyze byFlow CytometryWashDFNIRPE MFICDetector AntibodiesRedEMFIABConcentration (pg/mL)Standard Curve4BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.
OPEN AND CONFIGURABLEBD CBA 30-plex assay5104Enhanced sensitivity assaysThe BD CBA Enhanced Sensitivity Flex Set assays provide anew level of detection compared to the traditionalBD CBA Flex Set assays. The Enhanced Sensitivity Flex Setscan detect as low as 0.274 pg/mL in a multiplexed assay.This breakthrough allows researchers to get quantitativeresults in samples that were previously below the level ofdetection. The system includes assays for cytokines involvedin inflammation and leucocyte differentiation, which can becombined to create a multiplexed panel. The system providesa reliable, flexible method for quantitative detection ofmultiple proteins in a single human or mouse sample.BD CBA Cell Signaling Flex Set AssaysThe benefits of BD CBA assays are extended to researchersinvestigating cell signaling pathways. The assays cover keysignaling molecules involved in B-cell and T-cell receptorsignaling, as well as other pathways in the immune responsesuch as signaling via growth factor receptors and MAPkinase signaling.BD CBA products: designed for easy and efficientmultiplexing Require no assay formulation regardless of plex size Deliver quantitative results from a single small volume sample Require less total time and less hands-on time compared withcompetitor bead-based immunoassays Offer automated sample acquisition and increased throughputwith the plate-based BD FACSArray bioanalyzer, BD FACS Universal Loader, BD High Throughput Sampler, orBD CSampler accessoryNIR-A10310210210310104510Red-AThe assays include a recombinant protein standard thatprovides an internal control as well as a means to generatea standard curve and subsequent quantitative analysis.The intuitive analysis software generates a numericalreadout in relative units/mL for each protein assayed,delivering the answers needed without additional steps.Low inter- and intra-assay CVs allow researchers to havegreater confidence in results.BD CBA Flex Sets Open and configurable bead-based reagents Measure up to 30 analytes simultaneously on a flowcytometer with 488-nm or 532-nm and 633-nm lasersAvailable assays include: Soluble protein assays for detection of human, mouse,or rat cytokines, chemokines, and growth factors, humanimmunoglobulins Enhanced sensitivity assays for cytokine detection 1.0 pg/mL Cell signaling assays for detection of phosphorylated cellsignaling proteinsBD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.5
Build a Multiplex Step-by-StepBD CBA Flex Sets make it easy to build a multiplex byfollowing five simple steps. The finished assay can beacquired on a variety of dual-laser flow cytometers andanalyzed using FCAP Array software.InstrumentReporter ParameterClustering ParametersBD FACSVerse flow cytometerPECBA Red and CBA NIRBD Accuri C6 flow cytometer*FL2FL4 (675/25) and FL3 (780/60)BD FACSArray bioanalyzerYellowRed and NIRBD FACSCanto II flow cytometerPEAPC and APC-Cy 7BD LSR II flow cytometerPEAPC and APC-Cy7BD FACSAria III cell sorterPEAPC and APC-Cy7BD FACSCalibur flow cytometerFL2FL4 and FL3FCAP Array Parameters for BD CBA Flex Sets*Requires the BD Accuri Selectable Laser Module (Cat. No. 653126)STEP 1: Choose from our menu of Human, Mouse, Rat,Enhanced Sensitivity, and Cell Signaling BD CBA FlexSet Assays.Each BD CBA Flex Set comes with capture beads, detectionreagent, and standards. Sufficient reagents are provided torun 100 tests including two standard curves. All assays areavailable off-the-shelf and ready for mixing. WithBD CBA Flex Sets you can get right to work—no customorders required.STEP 2: Choose a 100 or 500 test size BD CBA Flex SetMaster Buffer Kit.Each BD CBA Flex Set Master Buffer Kit contains all the assayreagents and instrument setup beads necessary for any sizemultiplex configured from compatible BD CBA Flex Sets. Thismeans that for running a single-plex assay, a 10-plex assay,or larger, the buffer reagents are optimized to performwith the customized mixture selected and yield the correctnumber of assay tests.Human PBMCs1000000Protein levels using a BD CBA Flex Setassay100000Concentration (pg/mL)Human peripheral blood mononuclear cells(PBMCs) were stimulated under two differentconditions. Supernatants were collected andmeasured in a BD CBA Flex Set assay (30 plex).100001000100106BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic i-CD3/CD28IL-1βIL-7IL-6IL-5IL-4IL-21
MAXIMUM FLEXIBILITYA(A)STEP 3: Perform the assay following the instructions in the(A)Master Buffer Kit manual.p-ERKCon EGF TNF PDGFHEK-293(B)Final ConcentrationFinal Concentration(units/mL)(units/mL)Con EGFSTEP 4: Acquire samples on a dual-laser flow cytometer.p-ERKBD CBA Flex Set reagents have been verified for performance1400.00on a number of BD dual-laser flow cytometry platforms.(B)STEP 5: Analyze data files using FCAP Array multiplexanalysis software.Use the intuitive FCAP Array software to plot standardcurves and calculate sample 0.00400.00200.000.00Con NIH-3T3EGF TNF PDGFCon EGFTNF PDGFTNF PDGFB293 Con 293 EGF 293 TNF 293 PDGFControl 3T3 EGF293 Con 293 EGF 293 TNF 293 PDGFControl 3T3 EGF3T3 TNF 3T3 PDGF3T3 TNF 3T3 PDGFAnalysis of phospho-ERK1/2 protein levels in HEK 293and NIH 3T3 cells in response to EGF, TNF, and PDGFstimulation.A shows the results of Western blot analysis.B shows the results of BD CBA Flex Set analysis.Data courtesy of Dr. Tony Pawson and Dr. Jay Park, Mount SinaiHospital,Toronto, Canada.*50*****4035SF130SF225SF320SF41510ES Human TNFES Human IL-10ES Human IL-8ES Human IL-6ES Human IL-550ES Human IL-1βSynovial fluid samples from autoimmunedonors were diluted 1:10 and screened inthe Human Enhanced Sensitivity Flex Setsystem (11-plex). Analytes with detectableamounts are shown in the graph. Mostsamples ( * ) had IL-6 or IL-8 levels thatwere greater than the top point on thestandard curve ( 200 pg/mL).Concentration (pg/mL)Protein levels using a BD CBAEnhanced Sensitivity Flex Set assay*45BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.7
BD CBA Kits: Consistent Results withRoutine PanelsBD CBA Kits provide preconfigured panels for ultimateease of use. These kits enable multiplex analysis of complexbiological samples on a flow cytometer. In contrast toBD CBA Flex Sets, the kits are preconfigured by functionalareas of biology (eg, Th1/Th2 or inflammatory cytokines) tomeasure up to seven analytes simultaneously using capturebeads that contain unique amounts of a single red dye.The unique spectral properties of this dye enable analysisof samples on flow cytometers that have a single 488-nmlaser or on dual-laser (488-nm or 532-nm and 633-nm) flowcytometers. Each kit comes complete with all of the buffersand reagents necessary to analyze 80 samples.InstrumentReporter ParameterClustering ParametersBD FACSVerse flow cytometerPECBA RedBD Accuri C6 flow cytometerFL2FL4 (675/25)BD FACSArray bioanalyzerYellowRedBD FACSCanto II flow cytometerPEAPCBD LSR II flow cytometerPEAPCBD FACSAria III cell sorterPEAPCBD FACSCalibur flow cytometer(single laser)FL2FL3BD FACSCalibur flow cytometer(dual laser)FL2FL4Consistent packages, pre-optimized, and ready to useEach assay has been stringently developed for ease of use,rapid data analysis, sensitivity, reproducibility, and quality.Each antibody pair used in the kits is evaluated fordynamic range, sensitivity, and parallel titration curves tonative biological samples. In addition, the assay diluent andwash buffers in each kit have been formulated to reducedetrimental effects of serum and plasma proteins on assayperformance.FCAP Array parameters for BD CBA Kits35234 46169 Preconfigured kits for consistent resultswith routine panels Available for functional areas of biologysuch as Th1, Th2, Th17, and inflammatorycytokines Measure up to seven analytessimultaneously Compatible with flow cytometers thathave a 488-nm laser8BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.Measurement of Th17 cultures usingthe BD CBA Th1/Th2/Th17 KitsCD4 human memory T cells isolatedfrom whole blood were stimulated withplate-bound anti-CD3 and soluble antiCD28 alone (blue) or in the presence ofrecombinant IL-23 (orange).459245004000Concentration (pg/mL)BD CBA 85943IL-20IFN-γTNF0IL-436IL-62630IL-17A0IL-10
CONSISTENCYMedian Fluorescence(MFI)Comparison of BD CBA Standards to the NIBSC/WHOInternational StandardsThe NIBSC protein standards are recognized by the WorldHealth Organization (WHO) as international biologicalstandards. They meet established requirements for accuracy,consistency, and stability. The NIBSC/WHO standards areassigned potency values in International Units (IU) ofbiological activity and nominal mass (ie, not absolute massvalues). Therefore they cannot be used to establish absoluteconcentrations for a cytokine preparation. However, thesestandards do provide a means to facilitate comparisons ofcytokine concentration values determined by experimentsconducted within different laboratories or methods.Titration curve comparing the BD CBA Human IFN-grecombinant standard to the NIBSC/WHOInternational Standard.100C3aC4a2550Count75C5a0The BD CBA Human Anaphylatoxin Kit (Cat. No. 561418) can be usedto quantitatively measure anaphylatoxin C3a, C4a, and C5a (bioactivecleavage products released from C3, C4, and C5 during complementactivation) protein levels in a single EDTA plasma or serum sample.The three bead populations are mixed together to form the beadarray, which is resolved in a red channel of a flow cytometer.NIBSC/WHOInternational StandardConcentration(pg/mL)The conversion factor is intended to be a guideline indicatingwhether a BD CBA assay over- or underestimates analyteconcentrations relative to the NIBSC/WHO standards.Researchers are advised to incorporate both sets ofstandards in their assays if they wish to derive data fromthe NIBSC/WHO standards. For a table of NIBSC conversionfactors, please see the BD CBA Product List included withthis brochure.BD CBA Human Anaphylatoxin KitHuman CBAStandard102103104105Red-ABD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.9
Simplify Analysis withFCAP Array v3.0 SoftwareWith FCAP Array software (v3.0), sample results areobtained just minutes after performing a CBA experiment.After following the assay protocol, simply collect your dataon a flow cytometer, export FCS data files, and then analyzethe data using FCAP Array software.PerformBD CBAassayAcquire dataon a flowcytometerAnalyze inFCAP ArraysoftwareFCAP Array v3.0 software Intuitive layout of samples and assignment to data files Standard dilution calculator for fast data entry Debris filtering and manual clustering options Results viewing by sample or analyte Graphical results display C ompatibility with data files from BD FACS brand flow cytometersand the BD Accuri C6 personal flow cytometer Automated workflow for the BD FACSVerse flow cytometer10BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.The intuitive linear workflow allows for analysis of aBD CBA assay on any PC workstation. Simply create a newexperiment or quickly recall plexes saved from previousexperiments. FCAP Array software enables completeanalysis of data, interpolation of sample concentrationsby comparison to a standard curve, and viewing of resultsin graphical or tabular format or in a formatted report.Raw data can also be exported for downstream analysis instatistical software.
ANALYSISUsers of the BD FACSVerse flow cytometer equipped withBD FACSuite software will enjoy many automated featuresfor even faster results. Based on keywords that are enteredprior to acquisition and read by FCAP Array software, analysisbegins as soon as the FCS data files are identified.These keywords include identification of sample type (eg,standard, test sample, or control), identification of a plexname that automatically assigns a plex if a match is foundin the FCAP Array plex library, and the sample dilutionfactor. This automated workflow also reads the SampleID and Experiment Name directly from the FCS data file,providing consistent identification of a sample throughoutthe entire workflow and verification that all data files arefrom a related experiment.FCAP Array v3.0 software can use data files exported inFCS 2.0 or 3.0 formats, providing analysis capability formajor BD instrument platforms that use BD FACSuite,BD FACSDiva , BD FACSArray , BD Accuri C6, orBD CellQuest Pro software. For FCS 3.0 data files,FCAP Array software is capable of properly displayingcompensated data on a log scale. In the event that thesoftware cannot identify gates for the bead clusters,intuitive gating tools are available within FCAP Arraysoftware to filter out debris or to create manual gates. Thisprovides significant time savings and ensures data integrity.BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.11
Additional Options to Enhance FlexibilityBD CBA Functional Beads to conjugate for uniquerequirementsBD CBA Flex Set Functional Beads are unconjugated beadsthat allow researchers to conjugate their own antibodyor protein of interest using sulfo-SMCC chemistry. Theconjugation procedure takes less than four hours usingcommon laboratory supplies along with the buffers in theBD CBA Functional Bead Conjugation Buffer Kit.This is an ideal platform for converting existing ELISAs intobead-based immunoassays that can be mixed with our portfolio of BD CBA Flex Set assays. The availability of multiplebead positions enables creation of novel multiplex panels,while the ability to prepare up to 1,000 tests in a singlereaction ensures consistency across a large number of tests.The procedure to conjugate a protein to a functional beadconsists of four major steps: Step 1: Bead Preparation Step 2: Protein Modification Step 3: Buffer Exchange to Remove Unreacted Components Step 4: Protein ConjugationThe estimated time to completion for the entire conjugationprocedure is 3.5 hours.Overview of the functional bead conjugation procedureUnlabeled -SMCCMaleimidePurified Antibody12BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.
ADDITIONAL OPTIONSAdvantages Simple conjugation procedure completed in lessthan 4 hours. Conjugation reaction requires less than 100 μg ofprotein at a concentration of 1 mg/mL. Multiple bead populations that can be usedindividually or multiplexed as needed. Ability to conjugate any protein molecule containinga free amino group to the beads. Compatible with a wide selection of flow cytometersfor ease of analysis. Specific reagents available for confirming the successof conjugation reactions. Supporting reagents and procedures available forperforming instrument setups and assays.Mean fluorescence intensity100001000100101110100100010000sIL-6R (pg/mL)Standard curve for a soluble IL-6receptor assay generated usingBD CBA Functional Bead E4following the conjugation procedurein the BD CBA Functional BeadConjugation Buffer Set manual.Data courtesy of Joseph Cannon and Gloria Sloan,Medical College of nFL3-HKLH103FL3-H104lgG102In the assay shown, spectrally distinct beads coated with keyholelimpet hemocyanin (KLH) or tetanus toxoid (TT) were incubatedwith a test serum to detect the presence of antibodies to the twoantigens. Incubating each sample with a mix of anti-IgG or anti-IgMdetection antibodies that are labeled with unique fluorochromesallows for determination of the isotype of anti-KLH or anti-TTantibodies. Additional beads coated with IgG or IgM serve aspositive controls, and a bead coated with myoglobin serves as thenegative control. The data shown demonstrates that the test serumis positive for TT and KLH specific IgG antibodies (A), but positiveonly for KLH specific IgM antibodies (B). All measurements aremade from one tube, saving time and sample.105A novel assay from the Custom Technology TeamB105A102103104105anti-human lgG PE FL2-H102103104105anti-human lgM FITC FL1-HBD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.13
SERVICESServices and SupportBD Biosciences instruments and reagents are backedby a world-class service and support organization withunmatched flow cytometry experience. Our integratedapproach combines high-content bioimaging and flowcytometry instrumentation with trusted, certified reagents,and advanced applications. The BD Biosciences tools enableour customers to discover more and obtain the mostcomplete picture of cell function, and at the same timeexperience improved workflow, ease of use, and optimalperformance.Researchers come to BD Biosciences not only for qualityproducts, but as a trusted lab partner. Our repository of indepth, up-to-date knowledge and experience is available tocustomers through comprehensive training, application andtechnical support, and expert field service.The Custom Technology TeamMobilizing technology for research applications requiresclose collaboration. The Custom Technology Team (CTT) atBD Biosciences works with customers to provide solutionsthrough custom reagents, panels, or assay protocols.Staffed by leading scientists with a breadth and depth ofscientific and technical expertise, the CTT will coordinatewith researchers to study the problem at hand, makerecommendations, and help implement the solutions. In thisway, BD Biosciences technical know-how is translated intopractical solutions that allow customers to focus on research.Technical application supportBD Biosciences technical applications support specialistsare available to provide field- or phone-based assistanceand advice. Expert in a diverse array of topics, BD technicalapplication specialists are well equipped to addresscustomer needs in both instrument and application support.Choose the BD CBA product for your needsBD CBA Flex Sets Open and configurable bead-basedreagents Measure up to 30 analytes simultaneouslyon a flow cytometer with 488-nm or532-nm and 633-nm lasersAvailable assays include: Soluble protein assays for detectionof human, mouse, or rat cytokines,chemokines and growth factors,human immunoglobulins Enhanced sesitivity assays for cytokinedetection 1.0 pg/mL Cell signaling assays for detection ofphosphorylated cell signaling proteins14BD CBA Kits Preconfigured kits for consistent resultswith routine panels Available for functional areas of biologysuch as Th1, Th2, Th17, and inflammatorycytokines Measure up to seven analytessimultaneously Compatible with flow cytometers thathave a 488-nm laserBD Functional Beads Unconjugated beads that allow researchersto conjugate their own antibody orprotein of interest Enable the creation of novel bead assaysBD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD flow cytometers are Class 1 Laser Products.Cy is a trademark of Amersham Biosciences Corp. Cy dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from AmershamBiosciences Corp only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.FCAP Array is a trademark of Soft Flow Hungary Ltd. 2012 Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, inany form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from BD Biosciences.BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 2012 BD23-11508-02To order products through Fisher Scientific, please add BDB to the beginning of each part number listed.www.fishersci.com/bdbiosciencesFor technical assistance, contact BD BiosciencesToll free: 877-232-8995Email: ResearchApplications@bd.comFor customer service, call 1-800-766-7000.To fax an order, use 1-800-926-1166.To order online: www.fishersci.com16BD flow cytometers are Class 1 Laser Products.For Research Use Only. Not for use in diagnostic or therapeutic procedures.BN1014126
BD flow cytometers are Class 1 Laser Products. 6 For Research Use Only. Not for use in diagnostic or therapeutic procedures. Instrument Reporter Parameter Clustering Parameters BD FACSVerse flow cytometer PE CBA Red and CBA NIR BD Accuri C6 flow cytometer* FL2 FL4 (675/25) and FL3 (780/60) BD FACSArray bioanalyzer Yellow Red and NIR
