title: 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.
authors.affiliation: Quanterix Corporation, 113 Hartwell Avenue, Lexington, MA 02421, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Rissin
authors.forename: Cheuk W
authors.initials: CW
authors.lastname: Kan
authors.forename: Linan
authors.initials: L
authors.lastname: Song
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Rivnak
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Fishburn
authors.forename: Qichao
authors.initials: Q
authors.lastname: Shao
authors.forename: Tomasz
authors.initials: T
authors.lastname: Piech
authors.forename: Evan P
authors.initials: EP
authors.lastname: Ferrell
authors.forename: Raymond E
authors.initials: RE
authors.lastname: Meyer
authors.forename: Todd G
authors.initials: TG
authors.lastname: Campbell
authors.forename: David R
authors.initials: DR
authors.lastname: Fournier
authors.forename: David C
authors.initials: DC
authors.lastname: Duffy
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation*; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D053582:Interleukin-1alpha / Q000097:blood; D053583:Interleukin-1beta / Q000097:blood; D015850:Interleukin-6 / Q000097:blood; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D061848:Optical Imaging; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000097:blood
chemical_list: D055910:Antibodies, Immobilized; D005456:Fluorescent Dyes; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha