B-Cell Research

Measurement of the types and amounts of immunoglobulins and cytokines that are secreted by B cells provides insight into the quality and quantity of their immune response—features that are also frequently altered in disease states.

Methods that allow multiplexed measurements are increasingly being used to measure immunoglobulins (Igs) and cytokines. In addition to the benefits of multiplex analysis, they have several unique advantages compared to classically used techniques.


BD CBA Assay Principle
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 (25 to 50 μL) 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.

FCAP Array™ analysis software gates on each 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.

BD Cytometric Bead Array: Multiplexed Quantitation

BD™ Cytometric Bead Array (CBA) is a bead-based immunoassay that can simultaneously quantify multiple analytes from the same sample. The BD CBA system uses antibody-coated beads to efficiently capture analytes, and flow cytometry for read-out.

The broad dynamic range of fluorescence detection, and multiplexed measurement, allow for small sample volume, fewer sample dilutions, and substantially less time to establish the value of an unknown, versus a conventional ELISA approach.

The BD CBA portfolio includes assays for measurement of a variety of soluble and intracellular proteins, including immunoglobulins and cytokines.

Visit bdbiosciences.com/cba for more information.

Table 1. Cytokine-producing B cells

Cell type Cytokines Secreted
B effector 1 (Be-1) IFN-γ, IL-12, TNF
B effector 2 (Be-2) IL-2, IL-4, IL-6, TNF
Regulatory B IL-10, TGF-β1
IgG Standard Curve
IgG Standard Curve

BD CBA Assays for Igs

The BD™ CBA Human Immunoglobulin Flex Set system provides ready-to-use reagents that serve as building blocks for the multiplexed quantitation of multiple Ig subclasses. Because the amount and type of antibody in a particular immune response can vary greatly, measurements of these parameters can provide insight into the response after immunization or vaccination, or serve as an indicator to assess immunoglobulin deficiency disorders. For example, BD CBA assays have been used to analyze the Igs and cytokines secreted by isolated B cells from HIV-infected individuals after CpG stimulation.1

BD CBA Assays for Cytokines

BD™ CBA Flex Sets for cytokines provide an open and configurable menu of bead-based reagents designed for easy and efficient multiplexing. The specificities include a wide range of human and mouse cytokines. Data comparing results using BD CBA Standards to the NIBSC/ WHO International Standards is available as a guideline, to facilitate comparisons of cytokine concentration values determined by different laboratories or methods.

Measuring which cytokines are secreted can also provide information about the activity of different functional B-cell subsets in a sample, such as B effector-1 or -2, or regulatory B cells (Bregs) secreting anti-inflammatory cytokines such as IL-10.2 BD CBA Flex Sets have been used to quantitate the secretion of several B-cell–related cytokines and chemokines after manipulation of exhausted tissue-like B-memory cells in HIV-infected individuals.3

  Total IgG IgM IgA
Donor 1 16.9 1.3 1.6
Donor 2 10.9 2.4 0.9
Donor 3 8.9 0.7 1.5
Donor 4 14.6 2.8 1.5
Donor 5 11.6 0.3 1.1
Expected Range 7–14 0.5–3.3 0.8–3.9
Quantitation of immunoglobulins in human serum samples
Quantitation of immunoglobulins in human serum samples

Serum from five donors was tested using BD™ CBA Human Immunoglobulin Flex Sets to analyze total IgG, IgM, and IgA. Data was acquired on a BD FACSArray™ flow cytometer and analyzed using FCAP Array software. Dot plots for a negative control (detector alone) and one of the samples are shown. The results calculated (in g/L) on the basis of standard curves are shown, as well as the expected range.


References

1. Malaspina A, Moir S, DiPoto AC, et al. CpG oligonucleotides enhance proliferative and effector responses of B cells in HIV infected individuals. J Immunol. 2008;181:1199-1206.

2. Lund FE. Cytokine-producing B lymphocytes–key regulators of immunity. Curr Opin Immunol. 2008;20:332-338.

3. Kardava L, Moir S, Wang W, et al. Attenuation of HIV-associated human B cell exhaustion by siRNA downregulation of inhibitory receptors. J Clin Invest. 2011;121:2614-2624.



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