The BD Cytometric Bead Array System (CBA)

1 The BD Cytometric Bead Array System (CBA)Table of ContentsIntroduction ..3A Multiplex Bead System You Can Count On ..3BD Cytometric Bead Array (CBA) Assay Overview ..4 Quality is Built in to Every BD CBA Product ..5 Standardization of BD CBA Standards to International (NIBSC) Standards ..9 Non-human Primate Cross-reactivity ..11BD FACSA rray Bioanalyzer is Ideal for BD CBA Applications ..12BD CBA Assay Troubleshooting Tips ..13 Product Listing .. and Windows are registered trademarks of Microsoft Research Use Only. Not for use in diagnostic or therapeutic applications are either tested in-house or reported in the literature. See Technical Data Sheets for does not include or carry any 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 Becton Dickinson and Company is strictly flow cytometers are class I (1) laser productsBD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company.

2 2004 BDA Multiplex Bead System You Can Count OnBD Biosciences has added a new twist to counting with beads. Werecognize the value of precious samples and data reproducibility,so we ve developed a technology to ensure both. The innovativeBD Cytometric Bead Array (CBA) technology allows forquantitative detection of multiple analytes in a single serum,plasma, tissue culture supernatant, or cell lysate sample. TheBD CBA System of assay kits, flow cytometers, and easy-to-usesoftware provides reproducible data and reliable performance thatyou can count on time and time otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for resale. Get multiple results from a singlesmall-volume sample Run one standard mixture to generatestandard curves for all your analytes Avoid artifacts associated withenzyme-dependent signal generation Achieve quantitative results with lesstime and labor Combine versatile flow cytometerswith ready-to-use kits and analysissoftware Automate sample acquisition andincrease throughput with the plate-based BD FACSA rray bioanalyzer orother BD flow cytometers equippedwith the high throughput sampler(HTS) the BD Cytometric Bead Array (CBA) System You Can:Flow cytometry is an analytical tool that allows for thediscrimination of different particles on the basis of size andcolor.

3 The BD CBA employs a series of particles with discretefluorescence intensities to simultaneously detect multiple solubleanalytes from a single serum, plasma, or tissue culturesupernatant sample. The BD CBA, combined with flow cytometry,creates a powerful multiple analyte (multiplex) assay System . TheBD CBA System uses the sensitivity of amplified fluorescencedetection by flow cytometry to measure soluble analytes with aparticle-based immunoassay. The combined advantages of thebroad dynamic range of fluorescence detection via flowcytometry, and the efficient capturing of analytes via suspendedparticles coated with distinct capture antibodies enable theBD CBA to use fewer sample dilutions to determine analyteconcentration in substantially less time (compared toconventional ELISA). The specific capture beads are mixed with the phycoerythrin (PE)conjugated detection antibodies and then incubated withrecombinant protein standards or test samples to form sandwichcomplexes. Following acquisition of sample data using the flowcytometer, the sample results are generated in graphical andtabular format using the BD CBA Analysis CBA Assay otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures.

4 Not for resale.+or+WASHINCUBATEANALYZEACQUIREBD CBAF igure 1. Typical BD CBA assay singleanalyte is shown for representational powerful BD Cytometric BeadArray (CBA) assays enable multiplexanalysis of complex biological sampleson a flow cytometer. Each assay has beenstringently developed for ease-of-use,broad instrument compatibility and rapiddata analysis, sensitivity, reproducibility,and with any complex assay, the ability ofthe novice user to become comfortablewith the assay is critical. In the BD CBAsystem, ease-of-use has been engineeredinto each and every kit. The standards foreach multiplex kit are provided togetherin a lyophilized format that yields a ready-to-use stock standard solution uponreconstitution. Each kit includes a pre-diluted detection reagent containing eachdetector antibody formulated at theiroptimal concentration. Even the assayprotocol has been designed to limithandling steps (Figure 1)and hands-ontime by allowing the researcher to add allkit reagents, samples, and/or standards totheir assay well or tube at the same assay is incubated and thencompleted with a short wash stepfollowed by sample analysis on a BD CBA assay is compatible withany flow cytometer that is equipped witha 488 nm laser and capable of detectingand distinguishing fluorescence emissionsat 576 and 670 nm.

5 In addition, analysissoftware capable of saving data in an file format is required. This includesall of the BD flow cytometry systems,however, it should be noted that stream-in-air instruments will yield lower assaysensitivities than other instruments. Thedata collected on a flow cytometer, oncesaved as an FCS file, can be rapidlyanalyzed using the BD CBA BD CBA software enables linearregression analysis of data files andextrapolation of sample values bycomparison against a known standardcurve. The software also has broadcompatibility as it is offered in both PCand Mac-compatible formats, requiringonly Microsoft Excel to run. With theBD CBA software , sample results areobtained within minutes of completing effort has been made toensure that each BD CBA kit has thehighest possible sensitivity and bestreproducibility. Each antibody pair used inthe kits is evaluated for dynamic range,sensitivity, and parallel titration curves tonative biological samples (Figures 2and 3).

6 In addition, the scientists atBD Biosciences have formulated the assaydiluent and wash buffers in each kit toreduce detrimental effects of serum andplasma proteins on assay this does not always yieldcomparable recovery results as singleassays (eg, ELISA), it does provide qualitymultiplex performance. Possibly the most important aspect of theBD CBA kits is the quality performancethat is built into each one. Usingtechniques and methods developed atBD Biosciences, a worldwide leader in theproduction and conjugation of antibodies,the reagents in a BD CBA kit arestringently tested during the developmentprocess. Each capture antibody used for acapture bead has been tested both beforeand after it is coupled to the , it is tested again when it iscombined with the other kit reagents tocomplete the manufacture of a batch ofkits. The same is true of the detectionantibodies, which are additionally testedbefore and after conjugated withR-phycoerythrin (R-PE) and again whenthey are formulated into a detectionreagent.

7 Finally, they are tested with theother reagents in a given kit batch afterthey are bottled. All of this effort is madeto ensure that the kit that is delivered tothe researcher meets their expectationsand provides equal performance everytime. Further, the quality of the BD CBAkits does not end with the release of theproduct. We are continually looking formethods to enhance kit performancethrough improved antibody pairs, newstandards formulations and optimizedbuffers for serum samples. Quality is Built into Every BD CBA Product5 Unless otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for IL-8 Figure 2a. Representative data generated using the BD CBA Human Inflammation bead fluorescence is Built into Every BD CBA otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for 2c. Representative data generated using the BD CBA Human Inflammation standard titration ControlStandards 80 pg/mlStandards 625 pg/mlStandards 5000 pg/mlFigures 2b.

8 Representative data generated using the BD CBA Human Inflammation data acquired using the BD CBA analysis IL-8 Human IL-1 Human IL-6 Human IL-10 Human TNFH uman IL-12p70 TNF110100100010000110100100010000 Concentration (pg/ml)10 Log ScaleSemiLog ScaleLinear ScaleLog Scale110100100010000110100100010000 Concentration (pg/ml)IL-101101001000100001101001000100 00 Concentration (pg/ml)IL-611010010001000011010010001000 0 Concentration (pg/ml)IL-811010010001000011010010001000 0 Concentration (pg/ml)IL-1 110100100010000110100100010000 Concentration (pg/ml)IL-12p707 Unless otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for 2e. Representative data generated using the BD CBA Human Inflammation CellQuest data for the detection of individual proteins demonstrating assay 2d. Representative data generated using the BD CBA Human Inflammation standard curves generated with the BD CBA is Built into Every BD CBA otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures.

9 Not for (pg/ml)Median Fluorescence CurveBD CBA Human MCP-1 standardActivated Human cell culture supernatant Figure curve comparing the BD CBA Human CCL2/MCP-1 recombinantstandard to the NIBSC/WHO International 3. Human Chemokine Kit II data for data showingthe parallel titration curves of the BD CBA Human MCP-1 standard (20-5000pg/ml) and an activated human cell culture supernatant. For presentationpurposes, the first dilution of the supernatant, 1:2, was plotted at 5000 point thereafter (1:4, 1:16, 1:64, 1:256, and 1:1024) were similarly (pg/ml)Median Fluorescence Intensity10100100010000110100100010000 Standard CurveBD CBA Human MCP-1 standardNIBSC Human MCP-1 StandardLaboratories throughout the world usedifferent bioassays and immunoassays tomeasure and report cytokine protein levelsthat are present in biological samples. Forthis reason, the availability ofinternational standard preparations ofproteins is essential to allow definitiveanalyses and comparison of results.

10 Theseprimary (aka, gold) standards arefrequently used to calibrate biologicalactivities and protein concentrationsbetween different secondary assaystandards used by gold standards provide a means todetermine relative concentrations ofunknown samples and an ability tocompare results between experiments andlaboratories. In order to support thecomparison of protein measurementsobtained with BD CBA kits,BD Biosciences evaluates the assayperformance of the standards provided invarious BD CBA kits with gold standardsfrom the National Institute for BiologicalStandards and Control (NIBSC). The NIBSC Human Protein Standards arerecognized by the World HealthOrganization (WHO) as InternationalBiological Standards. They meetestablished requirements for accuracy,consistency and NIBSC/WHO standards are assignedpotency values in International Units (IU)of biological activity and nominal mass(ie, not absolute mass values) for purposesof bioassay and immunoassaydeterminations. These InternationalStandards are not intended to be used assamples of purified , they cannot be used toestablish absolute concentrations orspecific activities for cytokinepreparations.