The quantitative analysis of cytokines and other analytes in plasma, serum, or cell lysate is becoming increasingly important in the assessment of disease management and progression, and cellular response to various stimuli, toxins, and genetic manipulations. This has resulted in a growing demand for more sensitive, rapid, and cost-effective technologies.
Multiplex immunoassays offer considerable advantages to researchers in that they reduce sample consumption, analysis time, and cost. Assay Designs (ADI) has launched a multiplex bead immunoassay platform (MultiBead) that capitalizes on the ability of flow cytometry to separate beads covalently coupled to antibodies based on size and fluorescent intensity (Figure 1).
Separate capture antibodies are immobilized on the surface of the different bead types, allowing multiple immunoassays to be performed on the same sample simultaneously. The antigen in the sample is captured by the antibody on the bead and detected using biotinylated antibodies.
Signal is generated in the assay by subsequent addition of streptavidin conjugated to phycoerythrin, which emits at a different wavelength than the fluorophore in the beads. By utilizing various bead sizes, each labeled with multiple fluorescence intensities, MultiBead assays are capable of simultaneously quantifying more than 30 analytes in a single sample.
In this article, we illustrate MultiBead technology through the use of ADI’s Heat Shock Protein/Chaperone 8-plex MultiBead Kit.
Heat shock proteins (HSPs) are ubiquitously expressed polypeptides that are increasingly expressed in response to a variety of metabolic insults. Increased HSP expression functions to protect the cell from damage associated with misfolded proteins and is often used as an early indicator of cell stress. HSPs serve as molecular chaperones, facilitating the synthesis and folding of proteins throughout the cell. In addition, HSPs have been shown to participate in protein assembly, secretion, trafficking, protein degradation, and the regulation of transcription factors and protein kinases.
The MultiBead kit is the first commercially available HSP/Chaperone multiplex immunoassay for flow cytometers. The assay utilizes monoclonal antibodies or antigen affinity purified polyclonal antibodies against eight different HSPs or HSP client proteins coupled to latex beads.