US 11,965,891 B2
Digital protein quantification
Jeremy Agresti, Richmond, CA (US); and Ronald Lebofsky, Kensington, CA (US)
Assigned to Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed by Bio-Rad Laboratories, Inc., Hercules, CA (US); and Bio-Rad Europe Gmbh, Basel (CH)
Filed on Dec. 29, 2016, as Appl. No. 15/393,445.
Claims priority of provisional application 62/273,249, filed on Dec. 30, 2015.
Prior Publication US 2017/0192013 A1, Jul. 6, 2017
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/68 (2006.01); B01J 19/00 (2006.01); C12Q 1/68 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/6869 (2018.01); C40B 20/04 (2006.01); C40B 70/00 (2006.01)
CPC G01N 33/6878 (2013.01) [B01J 19/0046 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6869 (2013.01); C40B 20/04 (2013.01); C40B 70/00 (2013.01)] 9 Claims
 
1. A method for generating a plurality of mixture partitions, the method comprising:
(i) providing a plurality of fixed and permeabilized single cells;
(ii) incubating the plurality of single cells with a library of at least 10 structurally distinct antibodies that have a specific binding affinity for target epitopes of cellular proteins, wherein each of the at least 10 antibodies is conjugated to a different epitope-specific oligonucleotide comprising a barcode sequence that identifies the target epitope, a unique molecular identifier (UMI), a first primer binding sequence, and a second primer binding sequence, wherein the incubating results in binding of the antibodies to their corresponding epitopes, if present, to form antibody-epitope complexes;
(iii) washing away unbound antibodies;
(iv) partitioning, after the incubating and washing steps, the plurality of fixed and permeabilized single cells into a plurality of mixture partitions; and
(v) providing a plurality of partition-specific oligonucleotides to each mixture partition, wherein each partition-specific oligonucleotide comprises a different barcode sequence and a primer binding sequence that is complementary to the first primer binding sequence of the epitope-specific oligonucleotides,
wherein each partition-specific oligonucleotide is covalently linked to a bead.