US 11,951,483 B2
Methods for conducting multiplexed assays
Eli N. Glezer, Del Mar, CA (US); Sudeep Kumar, Basking Ridge, NJ (US); Pankaj Oberoi, Rockville, MD (US); George Sigal, Rockville, MD (US); and Michael Tsionsky, Derwood, MD (US)
Assigned to MESO SCALE TECHNOLOGIES, LLC., Rockville, MD (US)
Filed by Meso Scale Technologies, LLC., Rockville, MD (US)
Filed on Mar. 29, 2021, as Appl. No. 17/215,962.
Application 17/215,962 is a division of application No. 16/227,081, filed on Dec. 20, 2018, granted, now 11,059,046.
Application 16/227,081 is a continuation of application No. 15/784,399, filed on Oct. 16, 2017, granted, now 10,201,812, issued on Feb. 12, 2019.
Application 15/784,399 is a continuation of application No. 14/847,761, filed on Sep. 8, 2015, abandoned.
Claims priority of provisional application 62/047,097, filed on Sep. 8, 2014.
Prior Publication US 2021/0291168 A1, Sep. 23, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/543 (2006.01); B01L 3/00 (2006.01); C12Q 1/6804 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/6876 (2018.01); G01N 33/68 (2006.01)
CPC B01L 3/50853 (2013.01) [C12Q 1/6804 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6876 (2013.01); G01N 33/54306 (2013.01); G01N 33/54353 (2013.01); G01N 33/6845 (2013.01); B01L 2300/021 (2013.01); B01L 2300/043 (2013.01); B01L 2300/0609 (2013.01); B01L 2300/0829 (2013.01); G01N 2458/10 (2013.01)] 26 Claims
 
1. A method of conducting a multiplexed binding assay for a plurality of analytes comprising a first analyte and a second analyte of interest comprising
(a) combining, in one or more steps, the following components:
(i) a sample comprising the plurality of analytes comprising the first analyte of interest and the second analyte of interest,
(ii) a first targeting agent immobilized on a first binding domain, wherein the first targeting agent comprises an oligonucleotide,
(iii) a first targeting agent complement connected to a first linking agent, wherein the first targeting agent complement comprises an oligonucleotide complementary to the first targeting agent oligonucleotide, forming a first oligonucleotide pair,
(iv) a first binding reagent connected to a first supplemental linking agent, wherein the first binding reagent is a binding partner of the first analyte,
(v) a second targeting agent immobilized on a second binding domain, wherein the second targeting agent comprises an oligonucleotide,
(vi) a second targeting agent complement connected to a second linking agent, wherein the second targeting agent complement comprises an oligonucleotide complementary to the second targeting agent oligonucleotide, forming a second oligonucleotide pair,
(vii) a second binding reagent connected to a second supplemental linking agent, wherein the second binding reagent is a binding partner of the second analyte, and
(viii) at least two copies of a bridging agent,
wherein,
a first copy of the bridging agent has a first binding site for the first linking agent and an additional binding site for the first supplemental linking agent, and a second copy of the bridging agent has a first binding site for the second linking agent and an additional binding site for the second supplemental linking agent;
(b) forming
(i) a first binding complex on the first binding domain comprising the first targeting agent, the first targeting agent complement, the first linking agent, the first copy of the bridging agent, the first supplemental linking agent, the first binding reagent and the first analyte, and
(ii) a second binding complex on the second binding domain comprising the second targeting agent, the second targeting agent complement, the second linking agent, the second copy of the bridging agent, the second supplemental linking agent, the second binding reagent and the second analyte, and
(c) measuring the amount of the first and second analytes on the first and second binding domains, respectively.