US 12,291,738 B2
Methods and/or use of oligonucleotide conjugates for assays and flow cytometry detections
David A. Schwartz, Encinitas, CA (US); Jimmy Williams, Santee, CA (US); Xinfang Zhao, San Diego, CA (US); Chunfang Zhao, San Diego, CA (US); William B. Busa, Bahama, NC (US); Stephen J. Kron, Oak Park, IL (US); and Amy Catherine Flor, Chicago, IL (US)
Assigned to The University of Chicago, Chicago, IL (US); and EMD Millipore Corporation, Burlington, MA (US)
Filed by The University of Chicago, Chicago, IL (US); and EMD Millipore Corporation, Burlington, MA (US)
Filed on Jul. 7, 2021, as Appl. No. 17/369,771.
Application 17/369,771 is a continuation of application No. 13/302,877, filed on Nov. 22, 2011, abandoned.
Claims priority of provisional application 61/483,186, filed on May 6, 2011.
Claims priority of provisional application 61/344,931, filed on Nov. 22, 2010.
Prior Publication US 2022/0170073 A1, Jun. 2, 2022
Int. Cl. C12Q 1/6804 (2018.01); C07H 21/00 (2006.01); C12Q 1/6834 (2018.01)
CPC C12Q 1/6804 (2013.01) [C07H 21/00 (2013.01); C12Q 1/6834 (2013.01)] 25 Claims
 
1. A live cell flow cytometry method for detecting a target of a sample comprising living cells, comprising:
i) mixing the sample comprising living cells with a molecular probe and a detectable component, wherein:
1) the molecular probe comprises a binding moiety conjugated to a first oligonucleotide sequence, wherein
a. the binding moiety comprises an antibody, an antibody-fragment, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a chelator, or combinations or derivatives thereof; and
b. the conjugation between the binding moiety and the first oligonucleotides comprises one or more covalent bond linkages selected from the group consisting of a hydrazine, an oxime, and a triazine; and
2) the detectable component is not immobilized on a solid support and comprises a signal generator conjugated to a second oligonucleotide having a sequence complementary to the sequence of the first oligonucleotide, wherein
a. the signal generator comprises a scaffold molecule further conjugated to more than one signal generating moieties, the scaffold molecule comprising a dendrimer, a polysaccharide, or combinations or derivatives thereof; and
b. the second oligonucleotide is directly conjugated to the scaffold molecule of the signal generator via one or more covalent bond linkages selected from the group consisting of a hydrazine, an oxime, and a triazine;
ii) binding the target in the sample with the binding moiety of the molecular probe;
iii) hybridizing the first oligonucleotide with the second oligonucleotide;
iv) reducing the potential for cross-talk by adding to the molecular probe and the detectable component an oligonucleotide comprising at least one of:
1) an unconjugated oligonucleotide having a sequence complementary to the sequence of the first oligonucleotide; and
2) an unconjugated oligonucleotide having a sequence complementary to the sequence of the second oligonucleotide; then
v) detecting a signal generated from the detectable component hybridized to the target-bound molecular probe using flow cytometry, wherein the hybridized detectable component is not immobilized on a solid support; and
vi) removing the hybridized detectable component from the bound target, wherein the removal is by a stripping and washing process.