Cytometric Bead Arrays: A Breakdown of the Technique

By Hidaya Aliouche, B.Sc.Reviewed by Megan Craig, M.Sc.

A cytometric bead array is a form of flow cytometry that allows users to quantify proteins simultaneously.

A Cytometric bead array offers several advantages over other quantifier assays such as enzyme-linked immunosorbent assay (ELISA) and western blot; namely, reduction in sample requirements and time to results.

Cytometric bead array also allows for the design and creation of assays that measure several analytes of diagnostic and research interest, including chemokines, inflammatory mediators, immunoglobulin isotypes, intracellular signaling molecules, adhesion molecules, apoptotic mediators, and antibodies.

The Relevance of Cytometric Bead Based Assays

The measurement of soluble mediators involved in immune regulation is a topic of great research interest. Immunologists and cell biologists have predominantly focused on the in vitro and in vivo measurement of several analytes and their associated receptors. These molecules form the basis of a cellular communication network that determines both normal immune functioning and the incidence of disease; therefore, assessing their relative levels is of great importance.

Moreover, directly quantifying target analytes is a critical determinant in the pharmaceutical industry. Several assays are capable of this function, a majority of which employ ELISA-based technology. This technology is effective when analyzing single analytes; however, increasingly, there is a great demand for the ability to quantify several types of analytes simultaneously and rapidly from a small sample size.

Sample size becomes a critical factor when the need to quantify multiple analytes is essential, for example, when monitoring disease. This demand has resulted in the enmeshment of ELISA-based methodology with flow cytometry to alleviate these issues. In particular, the use of fluorescently labeled microspheres has been essential.

Fluorescent Labelling and Cytometric Bead Assay Procedure

The basis of cytometric bead assay is the use of a specific protein-capturing antibody conjugated to a bead surface. Each antibody is associated with a specific fluorescent intensity, which provides a method of capturing a soluble analyte or set of analytes with beads of known size and fluorescence. This enables the detection of analytes using flow cytometry.

Detection is based on the formation of sandwich complexes. Sandwich complexes are comprised of capture bead + detection reagent + analyte. In this case, the capture bead and detection reagent are incubated with an unknown sample that contains a mix of several analytes, which enable the sandwich complexes to be formed. The formation of these sandwich complexes is measured using flow cytometry; this enables particles with fluorescent characteristics of both the bead and the detector to be identified.

Bead-based technologies can also be used in which populations of beads are identified by one type of fluorescence, and the analyte-dependant signal is produced by detection reagents that carry a second type of fluorescent signal.

A laser beam is used to stimulate the fluorescently encoded beads or microspheres, passing each of the beads individually through a flow cell, and recording the signal that is emitted; the immunoassay signal is produced through the binding of fluorescent conjugates.

Each of these beads is differentiated by light scatter characteristics; they have their FL3 (specific fluorescence) intensity (size). The analytes are covalently coupled by the use of a detection antibody with specific wavelength emission.

Specific emission fluorescence in the detection antibody is measured by the FL2 fluorescent signal on the appropriate bead.

The fluorescent signal produced is proportional to the quantity of bound analyte. To determine the concentration of each analyte in the test sample, established calibration curves are used using dedicated cytometric bead assay analysis software.

The design of cytometric bead assays enables detection of a variety of analytes and enables a high throughput means of detection, which improves sensitivity and decreases the sample required for analysis. This process can be simplified using magnetic microspheres, as these functional fluorescent microspheres confer target selectivity and minimize background interference. Using this technique, a signal can be read selectively by employing a certain fluorescent channel setting, which makes it particularly sensitive to small molecules.

The Advantages of Cytometric Bead Assays Over ELISA

The advantage of cytometric bead assays over ELISA can be attributed to the broad dynamic range of fluorescence detection via flow cytometry, which allows efficient capturing of analytes using suspended particles. This subsequently allows besides symmetric bead assay to quantify the concentration of an unknown analyte at a markedly faster rate, using fewer sample dilutions as compared to conventional ELISA:

• The sample size is ~1/X^th of that used in conventional ELISA assay; where X denotes the number of simultaneously quantified analytes from a single sample.
• A single set of diluted standards is used to produce a standard curve for each analyte.
• The time to results in cytometric bead assays is substantially faster in comparison to ELISA.
• It allows for multiple detection events in sample volumes that are below the minimum threshold required for traditional immunoassays: assays are performed in a single reaction; by comparison, several ELISAs, each of which is specific for one analyte in question must be conducted which subsequently requires a proportional fold increase in the amount of sample required to conduct them all.
• Cytometric bead assays can be performed on a clinical flow cytometer already installed in the laboratory.

The Applications of Cytometric Bead Assays

Alongside biological detection, particularly in the fields of immunology to analyze homeostatic and disease states. In this context, cytometric bead assays are used to detect chemokines, immunoglobulin isotypes, inflammatory mediators, intracellular signaling molecules, adhesion molecules, mediators of apoptosis, and antibodies. Alongside this, cytometric bead assays have been employed in the field of food and agriculture.