The Cytometric Bead Array System

Rudolf Varro, Roy Chen, Homero Sepulveda, and John Apgar Summary

Analytical cytometry has significant potential beyond cellular analysis. The inherent capability of flow cytometers to efficiently discriminate between uniformly sized particles based on their intrinsic properties provides the foundation for multiplex bead assays. The technology can be exploited in designing immunoassays, Western blot-like antibody assays, and nucleic acid hybridization assays. This chapter focuses on immunoassay applications. The multiplex bead assays have recently evolved as a new and increasingly popular area for flow cytometry, becoming a good alternative to enzyme-linked immunosorbent assay for efficient evaluation of panels of analytes. This chapter provides detailed information about two bead platforms, the BDTM Cytometric Bead Array kits and the BD Cytometric Bead Array Flex Set Assays.

Key Words: CBA; multiplex bead immunoassays; preconfigured kits; Flex-Set assays; soluble proteins; cell signaling proteins; cell lysates.

1. Introduction

Bead-based, flow cytometric immunoassays have the ability to simultaneously and quantitatively measure multiple antigens or antibodies in a small volume of biological fluids. The flow cytometers support a broad dynamic assay range for the multiplex assays. The technology may be utilized to analyze the networks of mediators expressed by cells during immune and inflammatory responses. Cytokines (1), chemokines (2), inflammatory mediators and their receptors, as well as immunoglobulins (3), are frequently described as target molecules for multiplex assays. In addition, the bead assays can be applied to the simultaneous analysis of cell signaling molecules (4) and follow various activation pathways.

The use of microspheres of different size or color is at the basis of constructing multiplexed immunoassays. Several analytes can be assayed in one tube using very small sample volumes. Three basic concepts were developed to establish multianalyte assays. Fulwyler (5) and McHugh (6) pioneered the flow multiplex area using beads of different sizes as carriers for antigens or antibodies. The beads carrying different analytes are differentiated by their different scatter characteristics. Binding of fluorescent detectors to the beads generate the immunoassay signal.

Beads of the same size may be identified and differentiated by one type of fluorescence, whereas the signal is generated by conjugates carrying a second type of fluorescent signal (7). This concept is useful to create low-complexity bead sets (8).

Combining two or more fluorescent indexing colors in individual beads further extended the usefulness of multiplex flow assays. The individual bead populations contain unique ratios of the incorporated dyes, and their differing fluorescent signature is used to identify a series of beads carrying different specificities. Bead indexing is achieved in multidimensional fluorescent space with the capability to reach up to 100 individual bead addresses in two dimensions (9). The immunoassay signal is generated by antibodies coupled to a fluorescent dye, which is not interfering with the bead indexing. New instruments were designed to exploit the capabilities of multiplex bead assays without requiring the complex expertise of flow cytometry. The LabMAP 100 by Luminex and the BD FACSArray Bioanalyzer (Fig. 1) by BD Biosciences both support the utilization of the familiar microplate format. Both of these instruments have two lasers, a red laser that excites the two dyes in the dual color beads, and a green laser that is used for exciting the bead-bound conjugates. The signal generator is most often phycoerythrin (PE), thus the green laser excitation maximizes signal-to-noise ratios.

Multiplex assays are well suited to demonstrate a pattern of antibody responses against infectious agents, thus providing a bead assay analog to the Western blot method. McHugh (10) presented a prototype Hepatitis C virus antibody assay for potential use in the blood bank. Faucher (11) has demonstrated the capability of the multiplex assays to detect HIV-1 antibodies from fresh plasma and from dried bloodspot specimens. Khan (12) utilized the multiplex format to construct a serological assay, which detected 10 highly prevalent mouse infectious pathogens in a single reaction.

Two types of cytometric bead array (CBA) assays were developed at BD BioSciences. The BD CBA kits contain all the necessary components to perform the assay. The BD CBA Flex Set assays, on the other hand, provide all the necessary components and protocols to create customized multiplex panels, allowing a mix-and-match strategy.

Cytometric Bead Array And

Fig. 1. BD Flow cytometers most often used with the cytometric bead array (CBA) assays. (A) BD FACSCalibur™ Flow Cytometer. The dual laser instrument is compatible with single color and dual color indexed CBA beads and assays constructed with these beads. (B) BD FACSArray™ bioanalyzer is a microplate-based instrument optimized for bead assays. The instrument utilizes a 635-nm red laser to index the dual color beads and a 532-nm green laser for exciting the signal generator phycoerythrin (PE) conjugate. Both single color and dual color indexed beads are compatible with the platform.

Fig. 1. BD Flow cytometers most often used with the cytometric bead array (CBA) assays. (A) BD FACSCalibur™ Flow Cytometer. The dual laser instrument is compatible with single color and dual color indexed CBA beads and assays constructed with these beads. (B) BD FACSArray™ bioanalyzer is a microplate-based instrument optimized for bead assays. The instrument utilizes a 635-nm red laser to index the dual color beads and a 532-nm green laser for exciting the signal generator phycoerythrin (PE) conjugate. Both single color and dual color indexed beads are compatible with the platform.

The CBA kits are utilizing beads, which are dyed with a single red fluorescent dye. Each different group of beads is labeled with a discrete level of fluorescent dye so that it can be distinguished by its mean fluorescence intensity (MFI) upon flow cytometric analysis. Beads within each group are covalently coupled with antibodies that can specifically capture a particular type of molecule present within biological fluids including serum, plasma, tissue culture super-natants, or cell lysates. The capture beads are mixed with PE-conjugated detection antibodies and standards, controls, or test samples to form sandwich complexes. Following acquisition of sample data using multicolor flow cytometry, the

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Table 1

List of Available BDTM CBA Kits

Human CBA kits

Kit name

Specificities in kit

Allergy/asthma mediator kit - I

IL-3, IL-4, IL-5, IL-7, IL-10, GM-CSF

Allergy/asthma mediator kit - II

IL-3, IL-4, GM-CSF, G-CSF, eotaxin

Anaphylatoxin

C4a, C3a, C5a

Apoptosis kit

Cleaved PARP, Bcl-2, active caspase-3

Chemokine kit - I

IL-8, RANTES, MIG, MCP-1, IP-10

Inflammation kit

IL-8, IL-1P, IL-6, IL-10, TNF-a, IL-12p70

Th1/Th2 cytokine kit - I

IL-2, IL-4, IL-5, IL-10, TNF-a, IFN-y

Th1/Th2 cytokine kit - II

IL-2, IL-4, IL-6, IL-10, TNF-a, IFN-y

Nonhuman primate CBA kits

Nonhuman primate Th1/Th2 kit

IL-2, IL-4, IL-5, IL-6, TNF-a, IFN-y

Mouse CBA kits

Immunoglobulin isotyping kit

IgG1, IgG2a, IgG2b ,IgG3 , IgA, IgM, IgE

Inflammation kit

IL-6, IL-10, MCP-1, IFN-y, TNF-a, IL-12p70

Th1/Th2 cytokine kit

IL-2, IL-4, IL-5, TNF, IFN-y

standard curves are compiled in graphic format and sample results are tabulated by the BD CBA software.

The assays are compatible with most of the BD flow cytometers, including FACScan, FACSCalibur, FACSVantage, FACSAria, FACSCanto, and FACSArray platforms. The performance characteristics of the multiplex assays, including sensitivity, spike recovery, dilution linearity, specificity, and intra- and interassay precision were determined for each kit.

The list of available CBA kits is summarized in Table 1. The immunoassay analysis is performed by a stand alone CBA software, which is compatible with both Apple Macintosh and PC computers. Figure 2 shows six standard curves constructed with data using the Th1/Th2 CBA panel.

The CBA kits are often utilized to profile immunological processes. Tear samples from nonallergic and allergic donors were tested and reduced levels of interleukin (IL)-10 was found in allergic donors as compared to nonallergics (13). This application was further developed by Sonoda (14). Inflammatory cytokine CBA panel was used to monitor IL-6, IL-8, and IL-10 levels in pediatric patients who underwent cardiopulmonary bypass procedure (15). A characteristic time-course of these cytokines was detected, with significant increase during the intraoperative phase and fast decrease in the postoperative phase.

Flex Set Dilution
Fig. 2. Standard curves for the BD™ CBA Human Th1/Th2 panel (interleukin [IL]-2, IL-4, IL-6, IL-10, tumor necrosis factor-a, and interferon-y), analyzed using BD CBA software.

Hodges evaluated plasma samples from neonates with confirmed bacterial infection using the inflammatory cytokine panel (16). IL-6, IL-10, and IL-12 showed significant increase in in utero infected cases, whereas infection acquired after birth did not result in increased cytokine expression. Lipopolysaccharide induced cytokine and chemokine expression was evaluated in human carotid lesions using inflammatory cytokine, Th1/Th2 cytokine, and chemokine panels (17). The chemokine panel was also useful in analyzing differences between uncomplicated influenza A and B cases and H5N1 influenza A infection (18).

The kits were also evaluated in combination with cellular assays. Orfao reported simultaneous detection of secreted and cell-bound Th1/Th2 cytokines (19) using the CBA system.

Specificities and number of analytes are fixed in the CBA kits. Flexibility in combining analytes was achieved with BD CBA Flex Set assays, which are based on dual-color dyed beads (Fig. 3B).

The Flex Set system provides an open and configurable menu of bead-based reagents designed to create multiplex assays to specified requirements. Antibody conjugation chemistry, pair optimization strategies, and direct PE-detection reagents assure consistent assay performance in complex biological samples. Each antibody pair is evaluated for dynamic range, sensitivity, and parallel titration to native biological samples. The assay diluent and wash buffers are formulated to reduce potential interferences of serum and plasma samples. Direct PE conjugates are used as detection reagents, this minimizes the risk of increased background caused by endogenous biotin. The Flex Sets are compatible with serum, plasma, tissue culture supernatant, or cell lysate samples. A list of the available specificities and their bead location is summarized in Table 2. The standards are provided as unit-dose pellets, which can be easily combined with any number of additional pelletized standards. Common assay components, such as setup particles, buffers, and diluents are combined in a master buffer kit. The assays may be run either in tubes or in microplate format.

The Flex Set reagents require the use of dual-laser flow cytometers capable of detecting and distinguishing fluorescence emissions at 576, 670, and >680 nm. The Flex Set assays are compatible with the dual laser FACSCalibur, LSRII, FACSAria, FACSVantage, FACSCanto instruments, and the FACSArray Bio-analyzer.

Data analysis of the acquired FCS 2.0 data files is performed using FCAP Array software, which automatically clusters dual color CBA beads. It is a template-based system, which allows the design of customized Flex Set assay at the computer workstation. Fig. 4 shows two standard curves, constructed for IL-6 and IL-8 by using the FCAP Array software. A 27-plex Flex Set assay, consisting of cytokines, chemokines, and other biological modifiers is demonstrated on Fig. 5.

Bead Position Cba

Fig. 3. Bead sets utilized in cytometric bead array (CBA) assays. (A) Single color fluorescent beads of graded red fluorescence. These uniform sized beads are discernable by their varying FL3 fluorescence. The single color beads are used in the BD™ CBA assay kits. (B) Dual color fluorescent beads. The 30 uniform sized beads are excited by the red laser and discernable in two dimensions by their individual red and near infrared fluorescence. The beads are utilized in the BD CBA Flex Set assays.

Fig. 3. Bead sets utilized in cytometric bead array (CBA) assays. (A) Single color fluorescent beads of graded red fluorescence. These uniform sized beads are discernable by their varying FL3 fluorescence. The single color beads are used in the BD™ CBA assay kits. (B) Dual color fluorescent beads. The 30 uniform sized beads are excited by the red laser and discernable in two dimensions by their individual red and near infrared fluorescence. The beads are utilized in the BD CBA Flex Set assays.

Fig. 6 shows the bead map of a 9-plex Flex Set assay which was configured to quantitatively measure T-cell activation in cell lysates. Fig. 7 demonstrates the kinetics of Jurkat cell activation with CD3/CD28 treatment. Flex set assays were used to evaluate IgM signaling enhancement through ZAP 70 in chronic lymphoid leukemia (20).

In this chapter, we summarize the specifics of both the fixed panels and the Flex Set CBA assays. We are focusing on the assay methodology, for detailed

Table 2

List of Available BDTM CBA Flex Set Assays

Human soluble protein flex sets

Analyte

Bead position

Analyte

Bead position

Angiogenin

C4

IL-8

A9

Eotaxin

C7

IL-9

B6

Fas ligand

C6

IL-10

B7

Basic FGF

C5

IL-12p70

E5

G-CSF

C8

IP-10

B5

GM-CSF

C9

LT-a

D7

IFN-y

E7

MCP-1

D8

IL-1ß

B4

MIG

E8

IL-2

A4

MIP-1a

B9

IL-3

D5

MIP-1b

E4

IL-4

A5

RANTES

D4

IL-5

A6

TNFa

D9

IL-6

A7

VEGF

B8

IL-7

A8

Mouse soluble protein flex sets

GM-CSF

B9

IL-9

B5

IFN-y

A4

IL-10

C4

IL-2

A5

IL-12p70

D7

IL-3

A8

IL-13

B8

IL-4

A7

KC

A9

IL-5

A6

MCP-1

B7

IL-6

B4

TNF-a

C8

Rat soluble protein flex sets

IFN-y

A6

IL10

A9

IL-4

B9

TNF-a

C8

IL-6

A9

Phosphorylation site-specific flex sets

Btk (Y551)a

D5

PLCg (Y783)

B7

ERK1/2(T202/Y204)

C4

RSK (T573)

D7

Itk (Y511)

C6

Stat1 (Y701)

C5

JNK1/2 (T183/Y185)

B5

Syk (Y352)

B9

eNOS (S1177)

C7

ZAP-70 (Y319)

B8

p38/MAPKinase (T180/Y182)

B6

(Continued)

CBA

133

Table 2 (Continued)

Total signaling protein flex sets

Stat1

D4

ZAP-70

B8

Syk

B9

"The assays are specific for the phosphorylation sites displayed in brackets for each cell signaling molecule.

"The assays are specific for the phosphorylation sites displayed in brackets for each cell signaling molecule.

Cytometric Bead Array
Fig. 4. CBA Flex Set standard curves for human interleukin (IL)-6 and IL-8. Data acquired on a BD FACSArray™ bioanalyzer and analyzed using the FCAP Array™ software.
Bead Position Cba
Fig. 5. A 27-plex Flex Set on a dual laser BD FACSCalibur™ instrument. (A) Dot plot representation of the 27-plex Flex Set. (B) List of the human specificities coupled to the different beads. Each unique bead position is defined as an alphanumeric address.

information on data handling using the BD CBA analysis software and FCAP Array software please refer to their respective user guides. Both of these documents are accessible at www.bdbiosciences.com. Additional technical information about FCAP Array software can be found at the Soft Flow website (www.softflow.com).

2. Materials

2.1. Th1/Th2 Human Cytokine Panel

This panel serves as an example of the ready-to-use CBA kits, which contain all the necessary assay components in a single package. Six bead populations

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2 3 4

5

#7 '

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9

8

FL4-H

Fig. 6. A 9-plex Flex Set for testing T-cell activation. Key to the bead specificities: (1) Itk, (2) ERK, (3) JNK, (4) P38, (5) PLCy, (6) ZAP70, (7) LAT, (8) c-Jun, (9) RSK.

with distinct fluorescence intensities have been coated with capture antibodies specific for IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-a, and interferon (IFN)-y. The six individual bead populations are mixed together during the assay preparation. Fig. 8 shows the FL3 histogram of the combined capture beads. The cytokine capture beads are combined with the PE-conjugated detection antibodies and then incubated with recombinant standards or test samples to form sandwich complexes. Following acquisition of sample data using the flow cytometer, the sample results are generated in graphical and tabular format using the BD CBA analysis software. The kit provides sufficient reagents for the quantitative analysis of 50 test samples and two standard curve sets.

2.1.1. Human Cytokine Capture Beads

There are 0.8 mL of each specific capture beads with discrete fluorescence intensity characteristics are supplied with the kit. The brightest bead in the kit is designated A1, the dimmest bead is A6. The beads are carrying the following specificities: A1= human IL-2, A2 = human IL-4, A3 = human IL-6, A4 = human IL-10, A5 = human TNF-a, A6 = human IFN-y. The mixed capture bead

Tnf Activity Assay Kit

Fig. 7. Kinetics of T-cell activation using anti-CD3/CD28. Jurkat cells were activated with anti-CD3 and anti-CD28 for different lengths of time and the cells were lysed. A 9-plex BD CBA assay using 10 ¡g of lysate was run measuring phosphorylated ERK, JNK, p38, PLCy, ZAP-70, Itk, LAT, c-Jun, and RSK. Using standard curves, concentration (U/mL) for each specificity was determined and the fold increase in activity was plotted.

TIME (MINUTES)

Fig. 7. Kinetics of T-cell activation using anti-CD3/CD28. Jurkat cells were activated with anti-CD3 and anti-CD28 for different lengths of time and the cells were lysed. A 9-plex BD CBA assay using 10 ¡g of lysate was run measuring phosphorylated ERK, JNK, p38, PLCy, ZAP-70, Itk, LAT, c-Jun, and RSK. Using standard curves, concentration (U/mL) for each specificity was determined and the fold increase in activity was plotted.

reagent is formulated to support a 50-^L/test volume. The beads need to be stored at 4°C, and cannot be frozen.

2.1.2. PE-detection Reagent

Four milliliters of the mixed detector reagent is provided, containing PE-labeled monoclonal antibodies against human IL-2, IL-4, IL-6, IL-10, TNF-a, and IFN-y. The combined detector reagent is formulated for use at 50 ¡¡L/test.

2.1.3. Cytokine Standards

Two vials of freeze-dried mixed standards are supplied with the kit, each vial containing a mixture of human recombinant IL-2, IL-4, IL-6, IL-10, TNF-a, and IFN-y. Each vial should be reconstituted in 0.2 mL of assay diluent to prepare a 10X bulk standard. The reconstituted 10X bulk standard contains 50 ng/mL of each recombinant human IL-2, IL-4, IL-6, IL-10, TNF-a, and IFN-y protein.

2.1.4. Instrument Setup Beads and Controls

One and a half-milliliter setup bead and 0.5 mL of each of a PE- and FITC-labeled controls are included in the kit. The PE control is a PE-conjugated antibody specific for the antigen coated on the setup bead, and formulated for

Fig. 8. FL3 histogram of the Th1/Th2 CBA kit beads. The individual bead peaks are labeled with the corresponding specificity.

use at 50 ¡¡L/test. The fluorescein (FITC) control is a FITC-conjugated antibody specific for the antigen coated on the setup bead, and formulated for use at 50 ¡L/test. The controls are used with the instrument setup bead to set the initial instrument compensation.

2.1.5. Buffers

One hundred-thirty milliliters wash buffer and 30 mL assay diluent is supplied in the kit. The wash buffer is phosphate-buffered saline (PBS) with protein and detergent additives. It is used for wash steps and to resuspend the washed beads for analysis. The assay diluent is a buffered protein solution used to reconstitute and dilute the human Th1/Th2 cytokine standards and to dilute test samples.

2.1.6. Instrumentation, Equipment, and Software Requirements

To run the CBA assay a flow cytometer equipped with a 488-nm laser capable of detecting and distinguishing fluorescence emissions at 576 and 670 nm (e.g., BD FACScan™ or BD FACSCalibur™ instruments) and BD CellQuest™ Software is required. Data analysis requires the BD CBA Software. Regular 12 x 75-mm sample acquisition tubes, such as BD Falcon™ tubes are used for the assay preparation and data acquisition.

2.2. Soluble Protein Flex Set Assays

The Soluble Protein Flex Set assay system allows combination of the available single bead assays (Table 2) to create multiplex panels, as required by the experimenter's needs. These assays are supporting human, mouse, and rat multiplex panels. Each individual capture bead population has a distinct and unique near-infra red and red fluorescence intensity signature. Each bead species are covalently coupled with a capture antibody specific for a given soluble protein. Each bead population is resolvable from the other bead species in the multiplex assay by their unique signature in the FL3 and FL4 channels of a FACSCalibur flow cytometer or in the near-infra red and red channels of a FACSArray Bioanalyzer. Each bead population is given an a-numeric position designation indicating its position relative to other beads in the BD CBA Flex Set system (Table 2, Fig. 3B). Beads with different positions can be combined in assays to create a multiplex assay. In a Flex Set assay the capture bead, PE-conjugated detection reagent, and standard or test samples are incubated together to form sandwich complexes. Following acquisition of sample data using the flow cytometer, the sample results are generated in a graphical and tabular format using the FCAP Array software.

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