Bead-Based Immunoassays

BD™ Cytometric Bead Array (CBA) solutions measure a variety of soluble and intracellular proteins, including cytokines, chemokines, growth factors, and phosphorylated cell signaling proteins using flow cytometry. BD CBA solutions enable analysis of up to 30 proteins using just 25 to 50 μL of sample in comparison to other methods such as ELISA and Western blot, which enable only one protein to be analyzed per sample. BD CBA solutions are available in preconfigured kits or in an open, configurable format for unique needs.

BD CBA solutions are available in preconfigured kits or in an open, configurable format for unique needs.

BD CBA Flex Sets provide an open and configurable method of detection, so that researchers can build their own multiplexes for protein quantitation as low as 10 pg/mL.

BD CBA Enhanced Sensitivity Flex Sets are capable of detecting cytokine concentrations as low as 0.274 pg/mL.

BD CBA Kits are preconfigured for achieving consistent results for routine panels.

BD CBA Multiplexed Bead-Based Immunoassays

BD™ CBA solutions are designed for multiplexed analysis, to provide more data using a single sample. Multiplexing is especially useful when only a small amount of sample is available, maximizing the number of proteins that can be analyzed. With BD CBA, up to 30 proteins can be analyzed using just 25 to 50 μL of sample. Other methods such as ELISA and Western blot require a similar amount of sample, but only one protein can be analyzed from the same volume. With the BD CBA Enhanced Sensitivity Flex Set system, it is possible to detect as low as 0.274 pg/mL in a multiplexed assay.

The BD CBA portfolio includes assays for measurement of a variety of soluble and intracellular proteins, including cytokines, chemokines, growth factors, and phosphorylated cell signaling proteins.

BD CBA assay principle

Each capture bead in the array has a unique fluorescence intensity and is coated with a capture antibody specific for a single analyte. 
A combination of different beads is mixed with a sample or standard and a mixture of detection antibodies that are conjugated to 
a reporter molecule (PE). Following incubation and subsequent washing, the samples are acquired on a flow cytometer.

The FCAP Array analysis software gates on each individual bead population and determines the median fluorescence intensity (MFI) for each analyte in the array. It generates a standard curve and performs interpolation of sample concentrations compared to the standard curve and generates an analysis report in tabular format.

Consistent Results with Routine Panels

BD™ CBA Kits provide preconfigured panels for ultimate ease of use. These kits enable multiplex analysis of complex biological samples on a flow cytometer. In contrast to BD CBA Flex Sets, the kits are preconfigured by functional areas of biology (eg, Th1/Th2 or inflammatory cytokines) to measure up to seven analytes simultaneously using capture beads that contain unique amounts of a single red dye. The unique spectral properties of this dye enable analysis of samples on flow cytometers that have a single 488-nm laser or on dual-laser (488-nm or 532-nm and 633-nm) flow cytometers. Each kit comes complete with all of the buffers and reagents necessary to analyze 80 samples.

Consistent packages, pre-optimized, and ready to use

Each 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 for dynamic range, sensitivity, and parallel titration curves to native biological samples. In addition, the assay diluent and wash buffers in each kit have been formulated to reduce detrimental effects of serum and plasma proteins on assay performance.

FCAP Array Parameters for BD CBA Kits

Instrument
Reporter Parameter
Clustering Parameters

BD FACSVerse flow cytometer

PE

CBA Red

BD Accuri C6 flow cytometer

FL2

FL4

BD FACSArray™ bioanalyzer

Yellow

Red

BD FACSCanto™ II flow cytometer

PE

APC

BD LSRFortessa™ flow cytometer

PE

APC

BD FACSAria™ III cell sorter

PE

APC

BD FACSCalibur™ flow cytometer (single laser)

FL2

FL3

BD FACSCalibur™ flow cytometer (dual laser)

FL2

FL4

Open and Configurable to Maximize Flexibility

The BD™ CBA Flex Set system provides an open and configurable menu of bead-based reagents designed to make it easy to create multiplex assays. Available specificities include soluble protein assays for detection of human, mouse, or rat cytokines, chemokines, and growth factors; human immunoglobulins; and cell signaling assays for the detection of phosphorylated cell signaling proteins. Up to 30 analytes can be measured simultaneously using the BD CBA Flex Set system on a flow cytometer equipped with 488-nm or 532-nm and 633-nm lasers. The assays have been formulated to be mixed into any size plex, and they are all sold individually to provide a highly flexible system. All products are available off-the-shelf so custom orders are not required. Each product area (Human Soluble Protein, Mouse/Rat Soluble Protein, and Human Immunoglobulin) has a unique Master Buffer Kit. All assays within each product area have been verified for performance in single-plex and in multiplexed scenarios to ensure consistent data.

Simple steps to building a BD CBA Flex Set multiplex

BD CBA Flex Sets make it easy to build a multiplex by following five simple steps. First choose the analytes to be measured and the corresponding buffer kit. Then follow the simple formulation instructions in the Master Buffer Kit manual. Assay components are all formulated at 1 µL per test for easy calculations, and the unique lyophilized standard pellets facilitate the combination of proteins to make the standards mix. The finished assay can be acquired on a variety of dual-laser flow cytometers and analyzed using FCAP Array™ software.

  1. Choose from our menu of Human, Mouse, Rat, and Cell Signaling BD CBA Flex Set Assays. Each BD CBA Flex Set comes with capture beads, detection reagent, and standards. Sufficient reagents are provided to run 100 tests including two standard curves. All assays are available off-the-shelf and ready for mixing. With BD CBA Flex Sets you can get right to work—no custom orders required.
  2. Choose a 100- or 500-test size BD CBA Flex Set Master Buffer Kit. Each BD CBA Flex Set Master Buffer Kit contains all the assay reagents and instrument setup beads necessary for any size multiplex configured from compatible BD CBA Flex Sets. This means that for running a single-plex assay, a 10-plex assay, or larger, the buffer reagents are optimized to perform with the customized mixture selected and yield the correct number of assay tests.
  3. Perform the assay following the instructions in the Master Buffer Kit manual.
  4. Acquire samples on a dual-laser flow cytometer. BD CBA Flex Set reagents have been verified for performance on a number of BD dual-laser flow cytometry platforms. The plate-based BD FACSArray bioanalyzer can be used to optimize assay throughput and workflow. Simply prepare samples and standards in a 96-well filter-bottom plate, launch the previously designed BD FACSArray template, load the plate, and experience truly hands-free sample acquisition.
  5. Analyze data files using FCAP Array multiplex analysis software. Use the intuitive, wizard-driven FCAP Array software to plot standard curves and calculate sample concentrations.

Instrument Reporter Parameters 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
BD FACSCanto™ II flow cytometer PE APC and APC-Cy™7
BD LSRFortessa™ flow cytometer PE APC and APC-Cy7
BD FACSAria™ III cell sorter PE APC and APC-Cy7
BD FACSCalibur™ flow cytometer FL2 FL4 and FL3

Detect <1 pg/mL of Cytokine

The BD™ Cytometric Bead Array (CBA) Enhanced Sensitivity Flex Set system, based on the established BD CBA Flex Set system and proprietary detection technology, provides a new level of detection compared to other multiplex assay systems. Most bead assays can detect down to 5–10 pg/mL. The Enhanced Sensitivity Flex Set assays can detect as low as 0.274 pg/mL—more than a 35-fold improvement. This breakthrough allows researchers to get quantitative results in samples (eg, cultures at early time points) that were previously below the level of detection. The system includes assays for 10 different cytokines involved in inflammation and leucocyte differentiation, which can be combined to create a multiplexed panel. The system provides a reliable, flexible method for quantitative detection of multiple proteins in a single serum, plasma, or tissue culture supernatant sample.

More data in less time from a single sample

Multiplexing is especially useful because it maximizes the number of proteins that can be analyzed in a small amount of sample. Analyze up to 10 proteins with just 25 to 50 μL of sample using the Enhanced Sensitivity Flex Set system. Other methods such as ELISA require a similar amount of sample, but can analyze only one protein from the same volume. Because such a small amount of sample is needed, multiplexing reduces hands-on time as well as time to results.

Flexible and ready to use

Based on the established BD CBA Flex Set system and its proprietary detection technology, Enhanced Sensitivity Flex Set assays are formulated for combination into any size plex. They are sold individually to provide a highly flexible system. A dedicated master buffer kit contains all the buffers needed for the assay as well as an optimized protocol for performing the assay.

Robust assay system

All assays have been characterized for performance in single-plex and multiplex scenarios to ensure consistent data. The antibody pair in each assay is evaluated for dynamic range, sensitivity, and parallel titration to native biological samples. Results from samples containing levels of analyte detectable using the standard BD CBA Flex Set assay correlated with the Enhanced Sensitivity Flex Set assay. The optimized assay protocol ensures low background and superior signal-to-noise while using a two-step detection protocol.

Instrument Reporter Parameters 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
BD FACSCanto™ II flow cytometer PE APC and APC-Cy™7
BD LSRFortessa™ flow cytometer PE APC and APC-Cy7
BD FACSAria™ III cell sorter PE APC and APC-Cy7
BD FACSCalibur™ flow cytometer FL2 FL4 and FL3

Additional Options to Enhance Flexibility

Use BD™ CBA Functional Beads to conjugate for unique requirements. BD CBA Flex Set Functional Beads are unconjugated beads that allow researchers to conjugate their own antibody or protein of interest using sulfo-SMCC chemistry. The conjugation procedure takes less than four hours using common laboratory supplies along with the buffers in the BD CBA Functional Bead Conjugation Buffer Kit. This is an ideal platform for converting existing ELISAs into bead-based immunoassays that can be mixed with our portfolio of BD CBA Flex Set assays. The availability of multiple bead positions enables creation of novel multiplex panels, while the ability to prepare up to 1,000 tests in a single reaction ensures consistency across a large number of tests.

Functional bead conjugation

The BD™ CBA Flex Set system provides an open and configurable menu of bead-based reagents designed to make it easy to create multiplex assays.

Available specificities include soluble protein assays for detection of human, mouse, or rat cytokines, chemokines, and growth factors; human immunoglobulins; and cell signaling assays for detection of phosphorylated cell signaling proteins. Up to 30 analytes can be measured simultaneously using the BD CBA Flex Set system on a flow cytometer equipped with 488-nm and 633-nm lasers.

Featured Products

BD™ CBA Kits provide preconfigured panels for ultimate ease of use and enable multiplex analysis of complex biological samples on a flow cytometer. In contrast to BD CBA Flex Sets, the kits are preconfigured by functional areas of biology (eg, Th1/Th2 or inflammatory cytokines) to measure up to seven analytes simultaneously using capture beads that contain unique amounts of a single red dye. The unique spectral properties of this dye enable analysis of samples on flow cytometers that have a single 488-nm laser or on dual-laser (488-nm and 633-nm) flow cytometers. Each kit comes complete with all of the buffers and reagents necessary to analyze 80 samples.

Featured Product

Featured Instrument

Featured Software

CBA: Bead Intensities

Representative Standard Curves (IL-12p70 and TNF)

Representative Standard Curves (IL-10 and IL-6)

Representative Standard Curves (IL-1β and IL-8)

Titration curve comparison BD CBA vs NIBSC/WHO

Titration curve comparing the BD CBA Human IFN-γ recombinant standard to the NIBSC/WHO International Standard.

Titration Curve Comparison - Large

NIBSC conversion factor summary for BD CBA Kit Standards

Comparison of BD CBA Standards to the NIBSC/WHO International Standards. Download and print a PDF of this document.

BD CBA 30-Plex Assay

Stimulated Human PBMCs

Phospho-ERK1/2 Protein Detection

Example of BD CBA Functional Bead Assay

Titration curve comparison BD CBA vs NIBSC/WHO

Titration curve comparing the BD CBA Human IFN-γ recombinant standard to the NIBSC/WHO International Standard.

Titration Curve Flex Set Comparison - Large

NIBSC conversion factor summary for BD CBA Flex Set Standards

Comparison of BD CBA Standards to the NIBSC/WHO International Standards. Download and print a PDF of this document.

Comparison of BD CBA Flex Set vs BD CBA Enhanced Sensitivity Flex Sets.

Comparison of BD? CBA Flex Set vs BD CBA Enhanced Sensitivity Flex Sets 2

Brochures

Frequently Asked Questions

Product Information Sheets

Product List

User's Guides and Manuals

Webinars

CBA Kits

BD Accuri™ C6 flow cytometer

BD FACSAria™ III cell sorter

BD FACSArray™ flow cytometer

BD FACSCalibur™ flow cytometer

BD FACSCanto™ II flow cytometer

BD FACSVerse™ flow cytometer

BD LSRFortessa™ flow cytometer

Flex Sets

BD Accuri™ C6 flow cytometer

BD FACSAria™ III cell sorter

BD FACSCalibur™ flow cytometer

BD FACSCanto™ II flow cytometer

BD FACSVerse™ flow cytometer

BD LSRFortessa™ flow cytometer


*Requires the BD Accuri Selectable Laser Module (Cat. No. 653126)

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 Amersham Biosciences 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.

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