Multiplex Cellular Assays Using Detectable Cell Barcodes

Abstract

We describe herein a cell-based multiplexing technique called detectable cell barcoding (DCB). In DCB, each individual sample is labeled with a different DCB signature that distinguishes each sample by one or both of detected intensity or type of detection characteristic. The samples are then combined and analyzed for a detectable characteristic of interest (e.g., presence of an analyte). By employing multiple distinct DCB labels at varying concentrations, one can perform multiplex analyses on up to hundreds or thousands (or more) of cell samples in a single reaction tube. DCB reduces reagent consumption by factors of 100-fold or more, significantly reduces data acquisition times and allows for stringent control sample analysis.

1 - 21 . (canceled) 
     
     
         22 . A kit comprising at least one detectable cell barcode (DCB) label in a form that is optimized to DCB label multiple cell samples according the method of claim  1 , wherein said at least one DCB label is pre-measured into aliquots, wherein each of said aliquots comprises a different amount of said at least one DCB label that will distinguishably DCB mark starting cell samples in a multiplex DCB assay. 
     
     
         23 . The kit of  claim 22 , wherein said at least one DCB label comprises a cell binding moiety. 
     
     
         24 . The kit of  claim 23 , wherein said cell binding moiety is selected from one or both of a covalent cell binding moiety and a non-covalent cell binding moiety. 
     
     
         25 . The kit of  claim 22 , wherein said at least one DCB label is in a liquid composition. 
     
     
         26 . The kit of  claim 22 , wherein said at least one DCB label is in a dry composition. 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . The kit of  claim 22 , wherein said aliquots are provided in a multi-well strip or multi-well plate. 
     
     
         30 . The kit of  claim 22 , wherein said kit comprises multiple distinct DCB labels. 
     
     
         31 . The kit of  claim 22 , wherein said kit further comprises reagents for performing a DCB assay, wherein said reagents are selected form one or more of from the group consisting of: buffers for reconstitution and/or dilution of said at least one DCB label, buffers for labeling cell samples with said at least one DCB label, wash buffers, DCB labeling control cells, DCB labeling control beads, fluorescent beads for flow cytometer calibration, one or more detectable binding agents specific for detection of a cellular characteristic of interest, analyte detection control reagents, and combinations thereof. 
     
     
         32 . The kit of  claim 31 , wherein said one or more detectable binding agents comprises an analyte-specific antibody. 
     
     
         33 . The kit of  claim 32 , wherein said analyte is a specific isoform of a protein. 
     
     
         34 . The kit of  claim 31 , wherein said analyte detection control reagents comprises one or more of: a non-specific isotype control antibody, a positive analyte control sample, a negative analyte control sample, and combinations thereof. 
     
     
         35 . The kit of  claim 22 , wherein said DCB label comprises a fluorescent dye. 
     
     
         36 . The kit of  claim 22 , wherein said DCB label comprises a mass label. 
     
     
         37 . The kit of  claim 24  wherein said covalent cell binding moiety is selected from the group consisting of: amine-reactive groups, thiol-reactive groups, hydroxyl reactive groups, aldehyde-reactive groups, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, thiols, aldehydes, hydrazides, azides, sulfonyl halides, and combinations thereof. 
     
     
         38 . The kit of  claim 30 , wherein each of said multiple distinct DCB labels can be distinguished from one another based on fluorescence emission wavelength, fluorescence polarization, fluorescence lifetime, light scatter, mass, molecular mass, or combinations thereof. 
     
     
         39 . The kit of  claim 31 , wherein said one or more detectable binding agents comprises a DNA binding agent. 
     
     
         40 . The kit of  claim 31 , wherein said one or more detectable binding agents comprises a detectable catalytic activity. 
     
     
         41 . The kit of  claim 33 , wherein said specific isoform is the phosphorylated isoform of a protein. 
     
     
         42 . The kit of  claim 30 , wherein said aliquots comprise ready-made combinations of said multiple distinct DCB labels that will distinguishably DCB mark starting cell samples in a multiplex DCB assay. 
     
     
         43 . The kit of  claim 22 , wherein said DCB label is pre-measured into three or more aliquots.