USRE49774EActiveUtility
Methods for conducting multiplexed assays
Est. expiryMar 11, 2033(~6.7 yrs left)· nominal 20-yr term from priority
B01L 3/50853C12Q 1/6804C12Q 1/6816C12Q 1/6876G01N 33/54306G01N 33/54353G01N 33/6845B01L 2300/021B01L 2300/043B01L 2300/0609B01L 2300/0829G01N 2458/10C12Q 2563/131G01N 33/532G01N 2470/04
79
PatentIndex Score
0
Cited by
156
References
20
Claims
Abstract
The invention relates to methods for conducting solid-phase binding assays. One example is an assay method having improved analyte specificity where specificity is limited by the presence of non-specific binding interactions.
Claims
exact text as granted — not AI-modifiedThe invention claimed is:
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.
2. The method of claim 1 , wherein (i) the first targeting agent and first targeting agent complement and (ii) the second targeting agent and second targeting agent complement each comprise a complementary oligonucleotide pair.
3. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
1
acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2)
3
agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6)
6
atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12)
11
cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22)
13
cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26)
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)
20
gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40)
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.
4. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
2
acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4)
4
aggttcaftgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8)
5
atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10)
8
cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16)
10
catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20)
12
cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24)
14
cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28)
15
ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30)
19
ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38)
23
ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46).
5. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
2
acgtcccagttg (SEQ ID NO: 3)
caactgggacgt (SEQ ID NO: 4)
4
aggttcaftgca (SEQ ID NO: 7)
tgcactgaacct (SEQ ID NO: 8)
5
atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10)
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)
12
cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24)
14
cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28)
23
ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46).
6. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
1
acatcggtagtt (SEQ ID NO: 1)
aactaccgatgt (SEQ ID NO: 2)
3
agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6)
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)
12
cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24)
17
gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34)
18
gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36)
23
ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46)
25
tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50).
7. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
5
atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10)
6
atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12)
10
catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20)
11
cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22)
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)
19
ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38)
20
gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40).
8. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and 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)
6
atcattaccacc (SEQ ID NO: 11)
ggtggtaatgat (SEQ ID NO: 12)
9
caggtgtccatt (SEQ ID NO: 17)
aatggacacctg (SEQ ID NO: 18)
11
cctacgatatac (SEQ ID NO: 21)
gtatatcgtagg (SEQ ID NO: 22)
12
cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24)
16
gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32)
20
gttgattctgtc (SEQ ID NO: 39)
gacagaatcaac (SEQ ID NO: 40).
9. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
3
agaagaagatcc (SEQ ID NO: 5)
ggatcttcttct (SEQ ID NO: 6)
5
atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10)
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)
13
cggtttgagata (SEQ ID NO: 25)
tatctcaaaccg (SEQ ID NO: 26)
16
gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32)
23
ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46)
25
tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50).
10. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and 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)
10
catccaatccag (SEQ ID NO: 19)
ctggattggatg (SEQ ID NO: 20)
14
cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28)
17
gccatagtctct (SEQ ID NO: 33)
agagactatggc (SEQ ID NO: 34)
18
gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36)
25
tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50).
11. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
5
atcaggatacgc (SEQ ID NO: 9)
gcgtatcctgat (SEQ ID NO: 10)
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)
12
cgaatgtagagt (SEQ ID NO: 23)
actctacattcg (SEQ ID NO: 24)
14
cttacaacgcca (SEQ ID NO: 27)
tggcgttgtaag (SEQ ID NO: 28)
15
ctttctcggcac (SEQ ID NO: 29)
gtgccgagaaag (SEQ ID NO: 30)
19
ggtcgtgtttca (SEQ ID NO: 37)
tgaaacacgacc (SEQ ID NO: 38)
23
ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46).
12. The method of claim 2 1, wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is first oligonucleotide pair and second oligonucleotide pair are different and selected from:
Pair
Sequence
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)
8
cagaggtcttaa (SEQ ID NO: 15)
ttaagacctctg (SEQ ID NO: 16)
16
gacataaagcga (SEQ ID NO: 31)
tcgctttatgtc (SEQ ID NO: 32)
18
gctaattcacca (SEQ ID NO: 35)
tggtgaattagc (SEQ ID NO: 36)
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).
13. The method of claim 1 , wherein the components combined in step (a) further comprise: a first detection reagent that binds the first analyte of interest, the first detection reagent comprising a first detectable label; and a second detection reagent that binds the second analyte of interest, the second detection reagent comprising a second detectable label; and wherein the measuring step (c) further comprises measuring the presence of the first and second detectable labels via optical absorbance, fluorescence, phosphorescence, chemiluminescence, electrochemiluminescence, light scattering, or magnetism.
14. The method of claim 13 , wherein the first and second detectable label is an electrochemiluminescent label and the measuring step (c) further comprises measuring an electrochemiluminescent signal and correlating the signal with an amount of first and second analyte in the sample.
15. The method of claim 1 , wherein the first and second binding domains are positioned on an electrode and the measuring step further comprises applying a voltage waveform to the electrode to generate electrochemiluminescence.
16. The method of claim 1 , wherein each of the first and second binding domains is an element of an array of binding domains.
17. The method of claim 16 , wherein the array is located within a well of a multi-well plate.
18. The method of claim 1 , wherein each of the first binding domain is positioned on a surface of a first particle and the second binding domains are each domain is positioned on a surface of one or more particles a second particle.
19. The method of claim 18 , wherein the particles first particle and second particle are coded to allow for identification of specific particles each particle and discrimination between the first and second binding domains.
20. The method of claim 1, wherein each of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 45 and 49 are modified with a linking agent comprising a thiol group and each of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 46 and 50 are modified with a linking agent comprising a biotin group.Cited by (0)
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