Novel Immunoassay Platform for Evaluating Multiple Analytes with Minimal Sample Volume!--h2>
Maria Dinkelmann, Ph.D.
John M. Casper, Ph.D.
Michael C. Mullenix, Ph.D.
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.
HeLa cells were either heat-shocked or stimulated with anisomycin (a protein synthesis inhibitor and activator of stress-activated protein kinases) to induce a stress response. Cells were grown to ~80% confluency at 37ºC, heat shocked at 43ºC for two hours, and returned to 37ºC for 18 hours. Alternatively, cells grown to confluency were treated with 1 µg/mL anisomycin for 15 minutes. Both heat-shocked and anisomycin-treated cells were harvested by scraping, lysed with Cell Lysis Buffer 3, and adjusted to a protein concentration of 1 mg/mL. The Accuri C6 Flow Cytometer System (Accuri Cytometers) was utilized to analyze tissue culture lysates with the Heat Shock Protein/Chaperone 8-Plex MultiBead Kit in 96-well filter plates, according to kit instructions.
Recombinant standards of known concentrations were used to generate standard curves utilizing MultiBead software. The average mean fluorescent intensities (MFIs) from duplicate wells are shown in Figure 2A (ng/mL) and in Figure 2B (units/mL) against which MFIs of cell lysates were plotted to calculate HSP protein concentration.
The expression levels were converted to the percent of control lysates (Figure 3). Heat shock resulted in increased expression of Hsp60 and Hsp70. Hsp70 is considered an early responder to cell stress and plays multiple protective roles as a molecular chaperone including aiding in protein folding, participating in disposal of defective proteins, and preventing apoptosis. Hsp60 is a mitochondrial chaperone that aids in the re-folding of damaged proteins. Anisomycin treatment resulted in increased expression of Hsp60 and Hsp27pS82, consistent with its role as a kinase activator.
Multiplex immunoassays offer considerable advantages to researchers in that they reduce sample consumption, decrease sample analysis time, and are cost effective. The MultiBead immunoassay is capable of simultaneously detecting large molecules (cytokines, chemokines, and heat shock proteins) and small molecules (eicosanoids) by combining both immunometric and competitive immunoassays in a bead assay format that is compatible with commercially available flow cytometers.
The Accuri C6 Flow Cytometer System has a wide dynamic range: the user can view more than 6 logs of data at a time, further increasing the range of fluorescent intensity and number of analytes that can be analyzed in a single run. Combining the MultiBead assay panel with the Accuri C6 offers a compelling combination of value and flexibility for both multiplex immunoassays and cellular analysis research applications.