Multiplexed single molecule immunoassays

Abstract

We have developed a method that enables the multiplexed detection of proteins based on counting single molecules. Paramagnetic beads were labeled with fluorescent dyes to create optically distinct subpopulations of beads, and antibodies to specific proteins were then immobilized to individual subpopulations. Mixtures of subpopulations of beads were then incubated with a sample, and specific proteins were captured on their specific beads; these proteins were then labeled with enzymes via immunocomplex formation. The beads were suspended in enzyme substrate, loaded into arrays of femtoliter wells--or Single Molecule Arrays (Simoa)--that were integrated into a microfluidic device (the Simoa disc). The wells were then sealed with oil, and imaged fluorescently to determine: a) the location and subpopulation identity of individual beads in the femtoliter wells, and b) the presence or absence of a single enzyme associated with each bead. The images were analyzed to determine the average enzyme per bead (AEB) for each bead subpopulation that provide a quantitative parameter for determining the concentration of each protein. We used this approach to simultaneously detect TNF-α, IL-6, IL-1α, and IL-1β in human plasma with single molecule resolution at subfemtomolar concentrations, i.e., 200- to 1000-fold more sensitive than current multiplexed immunoassays. The simultaneous, specific, and sensitive measurement of several proteins using multiplexed digital ELISA could enable more reliable diagnoses of disease.

The Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing

“…Multiplexing Simoa digital immunoassays has been described. 26 At the instrument level, multiplexing involves the same process described above for singleplex assays. The ability to multiplex with Simoa technology rests with the ability to identify which beads loaded onto the array correspond to each assay.…”

“…To accomplish this, image analysis software was developed that first creates a "mask" of the array to define well positioning and boundaries for interrogation. 26 The well mask is then applied to the fluorescence images of the array to determine the presence of beads and enzymes within the wells. For the fluorescence images of the array, histograms of fluorescence intensity are generated for the bead population or bead subpopulations when multiplexing.…”

Emerging Technologies to Increase Ligand Binding Assay Sensitivity

“…The Simoa™ platform is capable of measuring up to ten different analytes simultaneously (multiplexing). This is achieved by using different dye linkers for each individual capture antibody (24). The array is then imaged using fluorescent detection at multiple wavelengths to identify subpopulations of different dyes and to determine the presence or absence of enzyme reporter labels.…”

Immunomagnetic nanoparticle-based assays for detection of biomarkers

“…Separately, Kan et al, and Rissin et al used a microarray system in which biomarkerbound magnetic particles are placed into each microwell for quantitative analysis. 45,46 Such methods provide a means to overcome the limitations of conventional microarrays, particularly with regards to the inflexibility of the system. Similarly, Bergo placed biomarker-bound magnetic particles into microwells, but also eluted the bound biomarkers to form discrete spots for microarray analysis via mass spectrometry.…”