US RE49,774 E1
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 Feb. 11, 2021, as Appl. No. 17/173,898.
Application 15/784,399 is a continuation of application No. 14/847,761, filed on Sep. 8, 2015, abandoned.
Application 17/173,898 is a reissue of application No. 15/784,399, filed on Oct. 16, 2017, granted, now 10,201,812, issued on Feb. 12, 2019.
Claims priority of provisional application 62/047,097, filed on Sep. 8, 2014.
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); B01L 3/00 (2006.01); C12Q 1/6804 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/6876 (2018.01); G01N 33/543 (2006.01); 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)] 19 Claims
 
1. A method of conducting a multiplexed binding assay for a plurality of analytes of interest comprising
(a) combining, in one or more steps, the following components:
(i) a sample comprising [ the plurality of analytes comprising ] a first analyte of interest and a 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 is a binding partner of the first targeting agent [ 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 is a binding partner of the second targeting agent [ 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) optionally, at least two copies of a bridging agent,
wherein,
if the bridging agent is omitted, each [ the first ] linking agent is a binding partner of the [ first ] supplemental linking agent [ and the second linking agent is a binding partner of the second supplementary linking agent] , or
if the bridging agent is included, the bridging agent has a first binding site for one of the linking agents [ the first linking agent or the second linking agent ] and an additional binding site for one of the supplemental linking agents [ the first supplemental linking agent or 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 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 binding reagent and the second analyte, and
(c) measuring an amount of the first and second analytes on the first and second binding domains, respectively
wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is [ first oligonucleotide pair and the second oligonucleotide pair are ] different and selected from:
 
Pair Sequence
 
 1 acatcggtagtt (SEQ ID NO: 1)
 
 aactaccgatgt (SEQ ID NO: 2)
 
 2 acgtcccagttg (SEQ ID NO: 3)
 
 caactgggacgt (SEQ ID NO: 4)
 
 3 agaagaagatcc (SEQ ID NO: 5)
 
 ggatcttcttct (SEQ ID NO: 6)
 
 4 aggttcaftgca (SEQ ID NO: 7)
 
 tgcactgaacct (SEQ ID NO: 8)
 
 5 atcaggatacgc (SEQ ID NO: 9)
 
 gcgtatcctgat (SEQ ID NO: 10)
 
 6 atcattaccacc (SEQ ID NO: 11)
 
 ggtggtaatgat (SEQ ID NO: 12)
 
 7 attaacgggagc (SEQ ID NO: 13)
 
 gctcccgttaat (SEQ ID NO: 14)
 
 8 cagaggtcttaa (SEQ ID NO: 15)
 
 ttaagacctctg (SEQ ID NO: 16)
 
 9 caggtgtccatt (SEQ ID NO: 17)
 
 aatggacacctg (SEQ ID NO: 18)
 
10 catccaatccag (SEQ ID NO: 19)
 
 ctggattggatg (SEQ ID NO: 20)
 
11 cctacgatatac (SEQ ID NO: 21)
 
 gtatatcgtagg (SEQ ID NO: 22)
 
12 cgaatgtagagt (SEQ ID NO: 23)
 
 actctacattcg (SEQ ID NO: 24)
 
13 cggtttgagata (SEQ ID NO: 25)
 
 tatctcaaaccg (SEQ ID NO: 26)
 
14 cttacaacgcca (SEQ ID NO: 27)
 
 tggcgttgtaag (SEQ ID NO: 28)
 
15 ctttctcggcac (SEQ ID NO: 29)
 
 gtgccgagaaag (SEQ ID NO: 30)
 
16 gacataaagcga (SEQ ID NO: 31)
 
 tcgctttatgtc (SEQ ID NO: 32)
 
17 gccatagtctct (SEQ ID NO: 33)
 
 agagactatggc (SEQ ID NO: 34)
 
18 gctaattcacca (SEQ ID NO: 35)
 
 tggtgaattagc (SEQ ID NO: 36)
 
19 ggtcgtgtttca (SEQ ID NO: 37)
 
 tgaaacacgacc (SEQ ID NO: 38)
 
20 gttgattctgtc (SEQ ID NO: 39)
 
 gacagaatcaac (SEQ ID NO: 40)
 
23 ttccacttaggg (SEQ ID NO: 45)
 
 ccctaagtggaa (SEQ ID NO: 46)
 
25 tttcccttgcta (SEQ ID NO: 49)
 
 tagcaagggaaa (SEQ ID NO: 50)
 
wherein each of SEQ ID NOs: 1, 5, 11, 21 and 25 are modified with a linking agent comprising a thiol group and each of SEQ ID NOs: 2, 6, 12, 22 and 26 are modified with a linking agent comprising a biotin group.