new bd cytometric bead array flex set system - … · new bd™ cytometric bead array flex set...

NEW BD™ Cytometric Bead Array Flex Set SystemThe easiest way to multiplex with beads

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

BD™ CBA Flex Sets - The Most Flexible Multiplex Bead Array System Available . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Customize Your BD™ CBA Flex Set Assay in a Few Easy Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

BD FACSArray™ Bioanalyzer and the BD™ CBA Flex Set Assay System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

A Perfect Combination for Superior Throughput . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

NEW FCAP Array™ Analysis Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

BD™ CBA Flex Sets - Ideal for Multiplex Quantitation of Phospho-Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

BD™ CBA Flex Set Assay Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

New Flexible System with Superior Quality and Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Product List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

2

For Research Use Only. Not for use in diagnostic or therapeutic procedures.All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.Purchase 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 prohibited. Macintosh and Mac are trademarks of Apple Computer, Inc., registered in the US and other countries.Microsoft and Windows are registered trademarks of Microsoft Corporation.BD™ flow cytometers are class I (1) laser products.FCAP Array™ is a registered trademark of Softflow, Inc.BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company.©2004-2005 Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrievalsystems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, orotherwise, without prior written permission from BD Biosciences.

BD™ CBA Flex Sets - The Most Flexible Multiplex Bead Array System Available

BD Biosciences has always provided the most reliable and complete multiplex bead array system.Now we’ve taken the performance you trust and the ease-of-use you rely on and made it ultimatelyflexible. In addition to your need for a flexible assay system, we also recognize the value ofyour precious samples and the importance of data reproducibility, so we’ve engineered the newBD™ Cytometric Bead Array (CBA) Flex Set system to maximize both.

Visit www.bdbiosciences.com/flexsets for more information.

With the BD™ CBA Flex Set System You Get:• Reagents that are the easiest and most efficient to multiplex

• A multiplex system that requires no assay formulation regardless of plex size

• Quantitative results from a single, small-volume sample

• Superior performance requiring less total time and less hands-on time than competitive bead-based immunoassays

• Results in less time than competitor assays

• Simple assay protocols yielding superior data

• Automated sample acquisition and increased throughput with the plate-based BD FACSArray™ bioanalyzer

• A complete system backed by the service and reliability you expect from BD Biosciences

Introduction

3Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale.All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.

Flow cytometry is an analytical tool that allows for thediscrimination of different particles on the basis of size andcolor. 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 recombinant proteinstandards or test samples, then incubated with phycoerythrin(PE)- conjugated detection antibodies to form sandwichcomplexes. Following acquisition of sample data using the flowcytometer, the sample results are generated using the new FCAPArray™ software.

The new BD CBA Flex Setsystem provides an open andconfigurable menu of bead-based reagents designed tobe the easiest method ofcreating multiplex assays.With our proprietaryconjugation chemistry, pairoptimization strategies, anddirect PE detection reagents,you can be assured ofconsistent and superior assayperformance in complexbiological samples.

Each antibody pair we develop is evaluated for dynamic range,sensitivity, and parallel titration to native biological samples. We have also specifically formulated the assay diluent and washbuffers for each assay type to reduce potentially detrimentaleffects on assay performance when using serum and plasmasamples. By avoiding a streptavidin-biotin-PE detection methodemployed by other assays, our direct PE detection reagentsminimize the risk of increased background often caused byendogenous biotin in serum and lysate samples. Thus, BD CBAFlex Sets provide a reliable and flexible method for quantitativedetection of multiple analytes in a single serum, plasma, tissueculture supernatant, or cell lysate sample.

For additional resources including instrument setup templates,please visit www.bdbiosciences.com/flexsets

Introduction (continued)

4Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale.

All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.

102 10 103 4 105

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Figure 1. BD CBA Flex Set Assay Protocol Schematic. Only a small representative sample of the available beads is shown.

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1) Choose from a menu of 100 test-size Human*,Mouse, Rat, and Cell Signaling BD™ CBA Flex SetAssays

Each BD™ CBA Flex Set comes with antibody-conjugated capturebeads, PE-conjugated detection reagents, and one or twostandards. The standards are provided as unit-dose pellets, whichcan be easily combined with any number of additional pelletizedstandards as you build larger and larger multiplexes.

* Please see technical data sheets for non-human primatereactivity information.

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 100 or 500tests of any size multiplex configured from compatible BD CBAFlex Sets. Whether you are running a single-plex, a 10-plex, orlarger, your buffer reagents are optimized to perform with yourcustomized mixture and yield the correct number of assay tests.

3) Install the NEW FCAP Array™ multiplex analysissoftware on your computer workstation

Using the intuitive, wizard-driven FCAP Array™ software, youcan design your BD™ CBA Flex Set assay at your workstation.Simply create a new template on demand, or quickly recalltemplates saved from previous experiments. For ease-of-use, youcan download a complete list of BD CBA Flex Sets and their beadposition identification labels (eg, A1, A2 …) fromwww.bdbiosciences.com/flexset. The list can be easily uploadedinto the FCAP Array software or you can add new bead positionson demand manually (more details about the FCAP Arraysoftware available on page 7).

4) Acquire samples on a dual-laser BD™ flowcytometer, such as the BD FACSArray™ bioanalyzer,then analyze your data using the FCAP Array™software

The BD™ CBA Flex Set reagents are compatible with dual-laserflow cytometers* equipped with a 488 nm and/or a 532 nm laser and a 635 laser capable of detecting and distinguishingfluorescence emission at 576 nm and 670 nm (off the 488 nmlaser) and 660 nm (off the 635 nm laser) such as a BD FACSCalibur™ or 576 nm (off the 532 nm laser) and 660 nmand >680 nm (off the 635 nm laser) such as a BD FACSArray™bioanalyzer. Additionally, acquisition software capable of saving data in an FCS 2.0 file format, such as BD CellQuest™ or BD FACSArray bioanalyzer software, is required. Assaythroughput can be optimized by using the plate-based BD FACSArray™ bioanalyzer. Simply prepare your samples and standards in a 96-well filter-bottom plate, launch yourpreviously designed FCAP Array™ template, load your plate, and experience truly hands-free sample acquisition.

* assays run on stream-in-air instruments may yield reduced sensitivities.

Customize Your BD™ CBA Flex Assay in a Few Easy Steps


Human Rodent Cell Lysate

Instrument setup information provided in the Master Buffer Kit instruction manuals.

A Perfect Combination for Superior Throughput Couple our BD™ CBA Flex Set assays with the plate-loader-equipped BD FACSArray™ bioanalyzer to significantly increaseyour lab’s throughput. The BD FACSArray bioanalyzer is a newflow cytometry platform for fast and sensitive high-contentanalysis of cells and proteins for cell biology, immunology, andproteomics applications.

The plate-based platform is perfect for multiplex bead assays,yet is also very flexible enabling complex cell-based applications.The BD FACSArray bioanalyzer is a uniquely flexible platformthat is easy-to-use, yet provides high performance. The two-laserplatform is a compact bench top unit that comes equipped witha fast microtiter-plate sampler and is capable of up to six-parameter detection.

BD FACSArray™ Bioanalyzer & the BD™ CBA Flex Set Assay System



With the flexible FCAP Array™ software, youcan easily recall a saved assay template or createa new one from scratch. After your template isdesigned, simply collect your BD™ CBA Flex Setdata on a flow cytometer, export FCS 2.0 datafiles, then analyze data using the FCAP Arraysoftware.

1) Create a new template or recall a saved one.

2) When creating a new template, customizeyour plex from a menu of BD™ CBA Flex Setspecificities manually or upload a completelist from www.bdbiosciences.com/flexset

The FCAP Array software enables complete analysis of data filesand extrapolation of sample values by comparison against astandard curve. Eight curve fitting models are offered includinglog-log or 4- and 5-parameter logistics. The software iscompatible with both PC (Windows 2000 and XP) and Macoperating systems (OS9.2, OSX - coming summer 2005). With theFCAP Array software, sample results are obtained within minutesof completing an experiment.

New FCAP Array™ Analysis Software


3) After your assay has been run and dataacquired, use the drop-down menu to selectthe flow cytometer your data was collectedon, then the software will display your beadcluster locations. Assign your bead labels tothe bead clusters with a simple double-click.

4) Select your curve-fitting algorithm.

New FCAP Array™ Analysis Software (continued)



5) View single standard curves or your raw data,and change curve-fitting models on demand.

6) Generate custom reports for everyexperiment.

New FCAP Array™ Analysis Software (continued)


Unlike conventional assays used to detect or measurephosphorylated proteins such as immunoprecipitation andWestern blotting, the BD™ CBA Flex Sets can provide sensitive,quantitative measurements of protein phosphorylation inconsiderably less time. In addition, the phosphorylation statusof multiple proteins can be measured in the same sample in aquantitative fashion (Figure 3 and Figure 4). The assays aredesigned to measure either phosphorylated or total specificitiesfrom a cell lysate. The specificity of the assays has been validatedby immunoprecipitation and Western blotting to ensure that onlythe protein of interest is detected (Figure 5). Test samples are runagainst a standard curve which is generated using dilutions of aphosphorylated recombinant standard. We have assessed thelinearity of the assays by making dilutions of activated cell lysatesand comparing them against the standard curve (Figure 2).In all cases the dilution curve of the sample is parallel to thestandard curve. Reproducibility experiments demonstrated a low%CV for intra-assay and inter-assay reproducibility plus thesensitivity of the assay is comparable to or better than aconventional Western blot.

BD™ CBA Flex Sets - Ideal for Multiplex Quantitation of Phospho-Proteins



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Figure 2. Jurkat cells activated with anti-CD3 and anti-CD28 andcrosslinked by anti-mouse Ig for 2 minutes. The reaction was stopped bythe addition of SDS (1% final) whereupon the samples were placed in aboiling water bath. A phosphorylated recombinant standard was used togenerate a standard curve. The upper panel shows the standard curveversus a titration of the activated Jurkat lysate. An important characteris-tic of the assay is that these two curves are parallel to each other. Thisensures linearity of the readings so that twice as much lysate will havetwice as many units/ml. The same concentrations of lysate were also runon an SDS-PAGE gel followed by immunoblotting with an anti-phospho-ZAP-70 antibody. Both a 30 second and a 10 minute exposure are shown.The BD CBA Flex Set assay is at least as sensitive as a Western blot. In thisparticular example, phosphorylation of ZAP-70 can be detected in a BD CBA Flex Set assay using lysate from less than 1,000 cells.

Figure 3. Kinetic analysis of T cell activation by anti-CD3/CD28. Jurkatcells were activated with anti-CD3 and anti-CD28 for different lengths oftime. Lysates were prepared as outlined in Figure 2. A 9-plex BD CBAassay using 10 µg of lysate was run measuring phosphorylated ERK, JNK,p38, PLCγ, ZAP-70, Itk, LAT, c-Jun, and RSK. Using standard curves,units/ml were determined for each specificity and the fold increase inactivity was plotted.

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Multiplex Quantitation of Phospho-Proteins (continued)


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Specificity Change % of ControlZAP-70 655 100Itk 77 100LAT 170 100PLCγ 272 100ERK 2946 100JNK 141 100p38 2398 100c-Jun 142 100RSK 385 100

Specificity Change % of ControlZAP-70 970 148Itk 61 79LAT 157 92PLCγ 125 46ERK 372 13JNK 92 65p38 1692 71c-Jun 195 137RSK 9 2

Specificity Change % of ControlZAP-70 -10 -2Itk -8 -10LAT -2 -1PLCγ 19 7ERK -2 0JNK 15 11p38 475 20c-Jun 32 23RSK 22 6

MEK Inhibitor(PD-98059)

Src Family Kinase Inhibitor(PP2)

Figure 4. Effect of inhibitors on T cell signaling. Jurkat cells were pre-incubated with either buffer, 200 µM PD-98059 (MEK inhibitor), or 10 µm PP2 (Srcfamily tyrosine kinase inhibitor) for 20 minutes before being activated with anti-CD3/CD28 for 2 minutes. In the left panel, a T cell signaling pathway isshown. The 9 phospho-specificities that were tested simultaneously using BD CBA Flex Set beads are circled. The table at the bottom of the panel showsthe median fluorescence intensity (MFI) units for each specificity. The middle panel shows the effects of pre-incubation with PD-98059. Since this com-pound inhibits MEK, only ERK and RSK should be affected. This is shown in gray on the pathway. However, although ERK and RSK are almost completelyinhibited, this compound does have effects on other signaling molecules. In the right panel, Jurkat cells were pre-incubated with PP2 which inhibits Srcfamily kinases such as Lck. This should shut down all of the signaling which is what is seen in the table.

Figure 5. Activation of MAP kinases by different stimuli. Different cell types were activated with different stimuli. Cell lysates were prepared asdescribed in Figure 2 and a 3-plex BD CBA Flex Set was run to measure phosphorylated MAP kinases (ERK, JNK, p38). The bottom panel shows a Westernblot of the same lysates using phospho-specific antibodies. Different stimuli have different patterns of activation and the results from the BD CBA FlexSet assays correlate well with the results from the Western blots.

New Flexible System with Superior Quality andReproducibility

The BD™ CBA Flex Set system has been developed to take theguesswork out of building multiplex assays. We’ve designed andformulated all of the assay reagents so that you can easily buildthe multiplex assay of your choosing from pre-configuredcomponents – all with the same superior assay performance youexpect from a BD CBA product. The BD CBA Flex Set systemprovides the easiest-to-use multiplex assays with superiorreproducibility ideal for large or long-term studies. In the tablesbelow you will find performance data as well as representativecomparisons to other, commercially available bead-basedmultiplex assay systems.

Sensitivity

The assay limits of detection are determined by averaging theaverage median fluorescence intensity of 20 replicates of thenegative control (0 pg/ml), and the corresponding concentrationat two standard deviations above the average. Representativeassay sensitivity of select BD™ CBA Flex Sets is shown inTable 1 below.

Recovery

Individual protein was spiked into various matrices at threedifferent levels within the assay range. The serum and plasmaused in these experiments was diluted 1:4 before addition of theprotein as recommended for each assay. The recovery at each levelwas averaged and the overall range of results is also shown. Eachassay below (Table 2) is shown as an example of the recoveryperformance for human, mouse, and rat BD™ CBA Flex Set assays.

BD™ CBA Flex Set Assay Performance



Phospho Btk(Y551) 0.6

Phospho eNOS (S1177) 1.3

Phospho ERK1/2 (T202/Y204) 6.6

Phospho Itk (Y511) 3.7

Phospho JNK1/2 (T183/Y185) 0.4

Phospho p38 (T180/Y182) 0.7

Phospho PLCγ (Y783) 1.1

Phospho Rsk (T573) 0.5

Phospho Stat1 (Y701) 2.1

Phospho Syk (Y352) 0.5

Phospho ZAP70 (Y319) 1.6

Total Stat1 0.6

Total Syk 2.3

Total ZAP-70 1.3

BD™ CBA FLEX SET SENSITIVITY (UNITS/ML)

Human IL-4 Media 97% 93 - 101%

Human IL-4 Serum* 91% 81 - 100%

Human IL-4 Plasma** 82% 79 - 86%

Human IFN-γ Media 60% 54 - 72%

Human IFN-γ Serum* 63% 47 - 87%

Human IFN-γ Plasma** 49% 40 - 62%

Mouse IL-4 Media 99% 83 - 124%

Mouse IL-4 Serum*** 29% 25 - 37%

Mouse IFN-γ Media 88% 84 - 95%

Mouse IFN-γ Serum*** 83% 77 - 94%

Rat IL-4 Media 79% 59 - 101%

Rat IL-4 Serum*** 62% 48 - 79%

Rat IFN-γ Media 42% 17 - 66%

Rat IFN-γ Serum*** 22% 16 - 28%

RECOVERY

BD™ CBA FLEX SET SAMPLE TYPE AVERAGE (%) RANGE (%)

Table 1. Sensitivity

Human IL-4 0.3

Human IFN-γ 0.3

Mouse IL-4 0.1

Mouse IFN-γ 0.3

Rat IL-4 0.7

Rat IFN-γ 7.8

BD™ CBA FLEX SET SENSITIVITY (PG/ML)

Table 2. Recovery

*Serum is pooled sera from 800 – 1,000 donors.**Plasma is pooled EDTA-treated plasma from at least 20 donors.***Mouse or rat serum was taken and tested individually.

Reproducibility

The precision of each assay was evaluated by testing thereproducibility of the assay to quantitate protein levels of samplescontaining three different levels of protein. The samples weretested either on a single experiment (10 replicates of each sample)to test the intra-assay reproducibility or on four separateexperiments by four independent operators each running thesamples in duplicate to test the inter-assay reproducibility.

The reproducibility of selected BD™ CBA Flex Sets is shownbelow (Table 3) as well as compared to competitor assays(Table 4).

BD™ CBA Flex Set Assay Performance (continued)


Phospho Btk(Y551) 5.7% 5 - 7% 8.0% 5 - 13%

Phospho eNOS (S1177) 9.0% 4 - 18% 5.7% 3 - 9%

Phospho ERK1/2 (T202/Y204) 9.0% 3 - 14% 3.7% 3 - 5%

Phospho Itk (Y511) 7.0% 8 - 12% 5.3% 4 - 8%

Phospho JNK1/2 (T183/Y185) 7.7% 6 - 10% 4.0% 2 - 6%

Phospho p38 (T180/Y182) 7.7% 6 - 9% 7.3% 6 - 9%

Phospho PLCγ (Y783) 12.3% 10 - 15% 4.3% 3 - 6%

Phospho Rsk (T573) 6.3% 5 - 9% 5.7% 3 - 7 %

Phospho Stat1 (Y701) 10.7% 7 - 16% 9.3% 6 - 15%

Phospho Syk (Y352) 7.7% 5 - 9% 9.0% 4 - 17%

Phospho ZAP70 (Y319) 5.7% 5 - 6% 12.7% 10 - 18%

Total Stat1 5.7% 4 - 8% 3.7% 2 - 6%

Total Syk 7.7% 6 - 10% 7.0% 4 - 11%

Total ZAP-70 9.3% 7 - 12% 4.7% 3 - 6%

Human IL-4 8.5% 7 - 10% 3.0% 3 - 3%

Human IFN-γ 6% 5 - 7% 4.7% 3 - 7%

Mouse IL-4 7% 5 - 10% 5.3% 2 - 8%

Mouse IFN-γ 4.7% 3 - 6% 3.7% 2 - 6%

Rat IL-4 5.3% 4 - 7% 3% 2 - 4%

Rat IFN-γ 9.7% 7 - 13% 4.3% 4 - 5%

INTER-ASSAY REPRODUCIBILITY INTRA-ASSAY REPRODUCIBILITY

BD™ CBA FLEX SET AVERAGE % CV RANGE (%) AVERAGE % CV RANGE (%)

Table 3. Reproducibility

Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV

Phospho ERK1/2 (T202/Y204) 9% 4% n/a n/a n/a n/a 35% 19%

Phospho JNK1/2 (T183/Y185) 8% 4% n/a n/a 12% 11% 78% 71%

Phospho p38 (T180/Y182) 8% 7% n/a n/a 18% 15% 52% 67%

Human IL-4 9% 3% 13% 7% 9% 8% 10% 6%

Human IFN-γ 6% 5% 12% 8% 17% 12% 16% 10%

Mouse IL-4 7% 5% 15% 5% 11% 9% 15% 7%

Mouse IFN-γ 5% 4% 20% 11% 34% 12% 12% 7%

INTER-ASSAY INTRA-ASSAY INTER-ASSAY INTRA-ASSAY INTER-ASSAY INTRA-ASSAY INTER-ASSAY INTRA-ASSAYREPRODUCIBILITY REPRODUCIBILITY REPRODUCIBILITY REPRODUCIBILITY REPRODUCIBILITY REPRODUCIBILITY REPRODUCIBILITY REPRODUCIBILITY

Table 4. Reproducibility vs Competitor Assays

BD™ CBA FLEX SETS COMPETITOR A COMPETITOR B COMPETITOR C

Linearity

Individual protein was spiked into various matrices, or cell lysateswere prepared, then the samples were serially diluted and tested inthe various assays. The recovery at each level was averaged andthe overall range of results is also shown. Each assay below isshown as an example of the recovery performance of human,mouse, and rat BD™ CBA Flex Set assays.

The linearity of selected BD CBA Flex Sets is shown below(Table 5) as well as compared to competitor assays (Table 6).




Phospho Btk(Y551) Cell lysate 118.7% 111 - 128%

Phospho eNOS (S1177) Cell lysate 103.7% 100 - 108%

Phospho ERK1/2 (T202/Y204) Cell lysate 109.0% 100 - 117%

Phospho Itk (Y511) Cell lysate 91.3% 88 - 94%

Phospho JNK1/2 (T183/Y185) Cell lysate 109.0% 105 - 112%

Phospho p38 (T180/Y182) Cell lysate 85.0% 75 - 93%

Phospho PLCγ (Y783) Cell lysate 116.0% 107 - 124%

Phospho Rsk (T573) Cell lysate 103.3% 94 - 113%

Phospho Stat1 (Y701) Cell lysate 86.3% 82 - 93%

Phospho Syk (Y352) Cell lysate 79.7% 72 - 86%

Phospho ZAP70 (Y319) Cell lysate 99.3% 99 - 100%

Total Stat1 Cell lysate 74.3% 67 - 81%

Total Syk Cell lysate 121.7% 106 - 133%

Total ZAP-70 Cell lysate 90.7% 82 - 103%

Human IL-4 Media 97% 91 - 101%

Human IL-4 Serum* 91% 81 - 100%

Human IL-4 Plasma** 82% 79 - 86%

Human IFN-γ Media 56% 53 - 60%

Human IFN-γ Serum* 56% 53 - 58%

Human IFN-γ Plasma** 68% 55 - 82%

Mouse IL-4 Media 94% 84 - 104%

Mouse IL-4 Serum*** 68% 56 - 79%

Mouse IFN-γ Media 90% 86 - 93%

Mouse IFN-γ Serum*** 86% 82 - 90%

Rat IL-4 Media 82% 78 - 90%

Rat IL-4 Serum*** 78% 74 - 84%

Rat IFN-γ Media 31% 28 - 34%

Rat IFN-γ Serum*** 19% 17 - 20%

LINEARITY

BD™ CBA FLEX SET SAMPLE TYPE AVG. (%) OF EXPECTED RANGE (%)

Table 5. Linearity

Cell lysates were from cells treated to induce protein phosphorylation (or leftuntreated for the total protein assays).*Serum is pooled sera from 800 – 1,000 donors.**Plasma is pooled EDTA-treated plasma from at least 20 donors.***Mouse or rat serum was taken and tested individually.

Human IL-4 Media 100% 95 - 105% 73% 72 - 73% 53% 50 - 55% 102% 99 - 105%

Human IL-4 Serum 79% 79 - 79% 93% 90 - 97% 75% 74 - 75% 119% 115 - 123%

Human IL-4 Plasma 80% 77 - 82% 92% 89 - 95% 77% 73 - 83% 95% 79 - 107%

Human IFN-γ Media 56% 53 - 60% 55% 54 - 56% 30% 26 - 37% 113% 102 - 124%

Human IFN-γ Serum 56% 53 - 58% 67% 64 - 71% 41% 39 - 43% 129% 121 - 139%

Human IFN-γ Plasma 68% 55 - 82% 56% 55 - 57% 40% 38 - 44% 123% 118 - 127%

Mouse IL-4 Media 94% 84 - 104% 241% 231 - 255% 241% 228 - 259% 301% 239 - 419%

Mouse IL-4 Serum 68% 56 - 79% 367% 284 - 428% 372% 313 - 430% 57% 36 - 80%

Mouse IFN-γ Media 90% 86 - 93% 135% 115 - 173% 62% 10 - 146% 751% 726 - 785%

Mouse IFN-γ Serum 86% 82 - 90% 102% 72 - 123% 31% 22 - 45% 217% 190 - 264%

AVG. % OF AVG. % OF AVG. % OF AVG. % OFSAMPLE EXPECTED RANGE (%) EXPECTED RANGE (%) EXPECTED RANGE (%) EXPECTED RANGE (%)

Table 6. Linearity vs Competitor Assays


Protein used in each spiked sample was from the BD CBA Flex Set assays, and the potency of that protein versus the competitor’s standard may affect results.

Assay Range

The range of each assay is determined by the levels of the assaystandard used on each experiment. The standard curve ranges foreach assay is listed below. It is possible that the assay will detectlevels of protein above or below the range of the standard curve.To increase confidence in the result obtained for a sample, it isrecommended that only sample dilutions that fall within thestandard curve range be used for quantitation of unknowns.

Bead Compatibility Matrix*

The table below shows the multiplexing compatibility of arepresentative sample of BD™ CBA Flex Sets. For additionalspecificities, please see individual datasheets or visitwww.bdbiosciences.com/flexsets



Phospho Btk (Y551)

Phospho eNOS (S1177)

Phospho ERK1/2 (T202/Y204)

Phospho Itk (Y511)

Phospho JNK1/2 (T183/Y185)

Phospho p38 (T180/Y182)

Phospho PLCγ (Y783)

Phospho Rsk (T573)

Phospho Stat1 (Y701)

Phospho Syk (Y352)

Phospho ZAP-70 (Y319)

BTK (Y551) ENOS ERK1/2 ITK (Y511) JNK1/2 P38 PLCγ (Y783) RSK (T573) STAT1 (Y701) SYK (Y352) ZAP70 (Y319)BD™ CBA FLEX SET (S1177) (T202/Y204) (T183/Y185) (T180/Y182)

Table 8. BD™ CBA Flex Set Assay Compatibility

ERK1/2 (T202/Y204) 3.9 – 1,000 Units/ml

Itk (Y511), Btk(Y551) 3.9 – 1,000 Units/ml

JNK1/2 (T183/Y185) 3.9 – 1,000 Units/ml

p38/MAPKinase (T180/Y182) 3.9 – 1,000 Units/ml

PLCγ (Y783) 3.9 – 1,000 Units/ml

Stat1 (Y701) 3.9 – 1,000 Units/ml

Syk (Y352) 3.9 – 1,000 Units/ml

ZAP-70 (Y319) 3.9 – 1,000 Units/ml

Total Stat1 3.9 – 1,000 Units/ml

Total Syk 3.9 – 1,000 Units/ml

Total ZAP-70 3.9 – 1,000 Units/ml

Human IL-4 20 – 5,000 pg/ml

Human IFN-γ 20 – 5,000 pg/ml

Mouse IL-4 20 – 5,000 pg/ml

Mouse IFN-γ 20 – 5,000 pg/ml

Rat IL-4 20 – 5,000 pg/ml

Rat IFN-γ 20 – 5,000 pg/ml

SPECIFICITY STANDARD CURVE RANGE

Table 7. Assay Range

Total Stat1

Total Syk

Total ZAP70

BD™ CBA FLEX SET TOTAL STAT1 TOTAL SYK TOTAL ZAP70

may be combined

may not be combined

*Total signaling protein flex sets may not be run in the same well as phospho-specific flex sets.

Time to Result

The time to complete BD™ CBA Flex Set assays was tracked andcompared to competitor assays. Results include preparation ofreagents for the experiment, sample acquisition, and data analysis.




01

001

0001

00001

00001000100101

2-LIh

4-LIh

5-LIh

6-LIh

8-LIh

b1-LIh

01-PIh

01-LIh

α-TLh

1-PCMh

FNTh

β1-PIMh

07p21-LIh

γNFIh

GIMh

BD™ CBA Flex Set 15-plex Multiplex

MFI

Concentration (pg/ml)

Figure 6. Representation Standard curves generated using a 15-plex of BD CBA Flex Sets.

No. Washes 0 9 9 11

No. Aspirations 1 12 13 11

No. Incubations 2/1* 3 3 3

Time to Result 4-5 hrs 10 hrs 10 hrs 4 hrs

Table 9. Soluble Protein Assays: Protocol Comparison and Time to Result


* 2 for Human assays and 1 for Mouse assays

No. Washes 0 n/a 9 11

No. Aspirations 1 n/a 13 11

No. Incubations 1 n/a 3 3

Time to Result 6 hrs n/a 10 hrs 19-22 hrs

Table 10. Cell Signaling Assays: Protocol Comparison and Time to Result


17

* Total signaling protein flex sets may not be run in the same assay well as phospho-specific flex sets.

Product List

Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale.All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.

BD FACSArray Bioanalyzer 340128

Instrumentation

DESCRIPTION CONTENTS CLONE APPS SIZE CAT. NO. PRICE

FCAP Array Software PC Compatible Software and User's Guide. Required for use with CD 338621BD CBA Flex Sets. Compatible with BD CBA kits.

BD CBA Kit Software Mac and PC compatible software and user’s guide. Compatible only CD 550065with BD CBA kit format

BD™ Cytometric Bead Array Flex SetsSoftware

DESCRIPTION CONTENTS SIZE CAT. NO. PRICE

Cell Signaling Master Buffer Kit Buffers and setup reagents required for use with BD CBA Cell Sufficient for 100 tests of any 558223Signaling Flex Sets size BD CBA Flex Set

Cell Signaling Master Buffer Kit Buffers and setup reagents required for use with BD CBA Cell Sufficient for 500 tests of any 558224Signaling Flex Sets size BD CBA Flex Set

Phospho-specific and Total Signaling Proteins Supporting Reagents*


Btk (Y551) Hu Capture Bead and Detection Reagents plus 1 standard D5 100 tests 558236

ERK1/2 (T202/Y204) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard C4 100 tests 558234

Itk (Y511) Hu, Ms Capture Bead and Detection Reagents plus 1 standard C6 100 tests 558230

JNK1/2 (T183/Y185) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B5 100 tests 558235

eNOS (S1177) Hu Capture Bead and Detection Reagents plus 1 standard C7 100 tests 558239

p38/MAPKinase (T180/Y182) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B6 100 tests 558233

PLCγ (Y783) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B7 100 tests 558228

RSK (T573) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard D7 100 tests 558240

Stat1 (Y701) Hu, Ms Capture Bead and Detection Reagents plus 1 standard C5 100 tests 558222

Syk (Y352) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B9 100 tests 558237

ZAP-70 (Y319) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B8 100 tests 558229

Phospho-specific Flex Sets*

DESCRIPTION REACT CONTENTS BEAD LOCATION SIZE CAT. NO. PRICE

Stat1 Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard D4 100 tests 558227

Syk Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B9 100 tests 558238

ZAP-70 Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B8 100 tests 558232

Total Signaling Protein Flex Sets*

DESCRIPTION REACT CONTENTS BEAD LOCATION SIZE CAT. NO. PRICE

Human Soluble Protein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Human Soluble Protein Flex Sets Sufficient for 100 tests of 558264any size BD CBA Flex Set

Human Soluble Protein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Human Soluble Protein Flex Sets Sufficient for 500 tests of 558265any size BD CBA Flex Set

Human Supporting Reagents

DESCRIPTION CONTENTS CLONE BEAD LOCATION SIZE CAT. NO. PRICE

Angiogenin Capture Bead and Detection Reagents plus 2 standards C4 100 tests Spring 2005

Soluble CD178 (sFas Ligand) Capture Bead and Detection Reagents plus 2 standards C6 100 tests Spring 2005

Eotaxin Capture Bead and Detection Reagents plus 2 standards C7 100 tests Spring 2005

basic FGF Capture Bead and Detection Reagents plus 2 standards C5 100 tests Spring 2005

G-CSF Capture Bead and Detection Reagents plus 2 standards C8 100 tests Spring 2005

GM-CSF Capture Bead and Detection Reagents plus 2 standards C9 100 tests Spring 2005

IFN-γ Capture Bead and Detection Reagents plus 2 standards E7 100 tests 558269/Spring 2005

IL-1β Capture Bead and Detection Reagents plus 2 standards B4 100 tests 558279/Spring 2005

IL-2 Capture Bead and Detection Reagents plus 2 standards A4 100 tests 558270/Spring 2005

IL-3 Capture Bead and Detection Reagents plus 2 standards D5 100 tests Spring 2005




IL-7 Capture Bead and Detection Reagents plus 2 standards A8 100 tests Spring 2005


IL-9 Capture Bead and Detection Reagents plus 2 standards B6 100 tests Spring 2005

IL-10 Capture Bead and Detection Reagents plus 2 standards B7 100 tests 558274/Spring 2005

IL-11 Capture Bead and Detection Reagents plus 2 standards E6 100 tests Spring 2005

IL-12p70 Capture Bead and Detection Reagents plus 2 standards E5 100 tests 558283/Spring 2005

IP-10 Capture Bead and Detection Reagents plus 2 standards B5 100 tests 558280/Spring 2005

LIF Capture Bead and Detection Reagents plus 2 standards D6 100 tests Spring 2005

LT-α Capture Bead and Detection Reagents plus 2 standards D7 100 tests 558284/Spring 2005

MCP-1 Capture Bead and Detection Reagents plus 2 standards D8 100 tests 558287/Spring 2005

MIG Capture Bead and Detection Reagents plus 2 standards E8 100 tests 558286/Spring 2005

MIP-1α Capture Bead and Detection Reagents plus 2 standards B9 100 tests Spring 2005

MIP-1β Capture Bead and Detection Reagents plus 2 standards E4 100 tests 558288/Spring 2005

Oncostatin M (OSM) Capture Bead and Detection Reagents plus 2 standards E6 100 tests Spring 2005

RANTES Capture Bead and Detection Reagents plus 2 standards D4 100 tests Spring 2005

TNF Capture Bead and Detection Reagents plus 2 standards D9 100 tests 558273/Spring 2005

VEGF Capture Bead and Detection Reagents plus 2 standards B8 100 tests Spring 2005

† Some specificities are reactive with Rhesus, Cynomolgus and Macaque non-human primate species

BD™ Cytometric Bead Array Flex Sets (continued)Human Flex Sets†


Mouse/Rat Soluble Protein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Mouse/ Sufficient for 100 tests of 558266Rat Soluble Protein Flex Sets any size BD CBA Flex Set

Mouse/Rat Soluble Protein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Mouse/ Sufficient for 500 tests of 558267Rat Soluble Protein Flex Sets any size BD CBA Flex Set

Mouse/Rat Supporting Reagents


18

Product List (continued)


GM-CSF Capture Bead and Detection Reagents plus 2 standards B9 100 tests Spring 2005

IFN-γ Capture Bead and Detection Reagents plus 2 standards A4 100 tests Spring 2005







IL-10 Capture Bead and Detection Reagents plus 2 standards C4 100 tests Spring 2005

IL-12p70 Capture Bead and Detection Reagents plus 2 standards D7 100 tests Spring 2005


Mouse Flex Sets


19

**IMPORTANT - Pre-configured Kits and Flex Sets MAY NOT be combined.

Product List (continued)


KC Capture Bead and Detection Reagents plus 2 standards A9 100 tests Spring 2005

MCP-1 Capture Bead and Detection Reagents plus 2 standards B7 100 tests Spring 2005

MIG Capture Bead and Detection Reagents plus 2 standards D9 100 tests Spring 2005

MIP-1α Capture Bead and Detection Reagents plus 2 standards C7 100 tests Spring 2005

MIP-1β Capture Bead and Detection Reagents plus 2 standards C9 100 tests Spring 2005

RANTES Capture Bead and Detection Reagents plus 2 standards D8 100 tests Spring 2005

TNF Capture Bead and Detection Reagents plus 2 standards C8 100 tests Spring 2005

BD™ Cytometric Bead Array Flex Sets (continued)Mouse Flex Sets


IFN-γ Capture Bead and Detection Reagents plus 2 standards A6 100 tests Spring 2005

IL-2 Capture Bead and Detection Reagents plus 2 standards E5 100 tests Spring 2005




TNF Capture Bead and Detection Reagents plus 2 standards C8 100 tests Spring 2005

Rat Flex Sets


Allergy/Asthma Mediator Kit – I IL-3, IL-4, IL-5, IL-7, IL-10, GM-CSF Appx 80 tests (samples plus standards) 558022

Allergy/Asthma Mediator Kit – II IL-3, IL-4, GM-CSF, G-CSF, Eotaxin Appx 80 tests (samples plus standards) 558023

Angiogenesis Kit IL-8, Basic FGF, VEGF, TNF, Angiogenin Appx 80 tests (samples plus standards) 558014

Apoptosis Kit Cleaved PARP, Bcl-2, Active Caspase-3 Appx 80 tests (samples plus standards) 557816

Chemokine Kit – I IL-8, RANTES, MIG, MCP-1, IP-10 Appx 80 tests (samples plus standards) 552990

Chemokine Kit – II IL-8, RANTES, MIP-1α, MIP-1β, MCP-1 Appx 80 tests (samples plus standards) 558015

Inflammation Kit IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70 Appx 80 tests (samples plus standards) 551811

Th1/Th2 Cytokine Kit – I IL-2, IL-4, IL-5, IL-10, TNF, IFN-γ Appx 80 tests (samples plus standards) 550749

Th1/Th2 Cytokine Kit – II IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ Appx 80 tests (samples plus standards) 551809

BD™ Cytometric Bead Array Ready-to-Use Kits**Human


Non-Human Primate Th1/Th2 Kit IL-2, IL-4, IL-5, IL-6, TNF, IFN-γ Appx 80 tests (samples plus standards) 557800

Non-Human Primate


Inflammation Kit IL-6, IL-10, MCP-1, IFN-γ , TNF, IL-12p70 Appx 80 tests (samples plus standards) 552364

Immunoglobulin Isotyping Kit Heavy and light chain isotypes of mouse IgG1, IgG2a , 100 tests 550026IgG2b ,IgG3 , IgA, IgM, IgE

Th1/Th2 Cytokine Kit IL-2, IL-4, IL-5, TNF, IFN-γ 50 tests 551287

Mouse


Human Inflammation Cytokine Standard IL-8, IL-1, IL-6, IL-10, TNF, IL-12p70 Lyophilized, 1 vial 552932

Human Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, IL-6, IL-10, TNF, IFN-γ Lyophilized, 1 vial 551810

Mouse Inflammation Cytokine Standard IL-6, IL-10, MCP-1, IFN-γ , TNF, IL-12p70 Lyophilized, 1 vial 620280

Mouse Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, TNF, IFN-γ Lyophilized, 1 vial 552967

Supporting Reagents


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