IP Library › Granted Patent US 10,201,812
Granted Patent B2
US 10,201,812 · App. 15/784,399 · Granted Feb 12, 2019

Methods for conducting multiplexed assays

Inventors: Eli N. Glezer (Del Mar, CA); Sudeep Kumar (Gaithersburg, MD); Pankaj Oberoi (Rockville, MD); George Sigal (Rockville, MD); Michael Tsionsky (Derwood, MD)
Assignee: Meso Scale Technologies, LLC.
B01L3/50853C12Q1/6804C12Q1/6816C12Q1/6876G01N33/54306G01N33/54353G01N33/6845B01L2300/021B01L2300/043B01L2300/0609B01L2300/0829G01N2458/10
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 10,201,812
App. No.
15/784,399
Granted
Feb 12, 2019
Kind
B2
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 (426)

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 a first analyte of interest and a second analyte of interest,

(ii) a first targeting agent immobilized on a first binding domain,

(iii) a first targeting agent complement connected to a linking agent, wherein the first targeting agent complement is a binding partner of the first targeting agent,

(iv) a first binding reagent connected to a 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,

(vi) a second targeting agent complement connected to a linking agent, wherein the second targeting agent complement is a binding partner of the second targeting agent,

(vii) a second binding reagent connected to a 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 linking agent is a binding partner of the supplemental linking agent, or

if the bridging agent is included, the bridging agent has a first binding site for one of the linking agents and an additional binding site for one of the supplemental linking agents;

(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 the 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 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 , wherein the complementary oligonucleotide pair positioned on each of the first and second binding domains is 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 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 and second binding domains are each positioned on a surface of one or more particles.

19. The method of claim 18 , wherein the particles are coded to allow for identification of specific particles and discrimination between the first and second binding domains.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 13, 2018
From: GLEZER, ELI N.; KUMAR, SUDEEP; OBEROI, PANKAJ; SIGAL, GEORGE; TSIONSKY, MICHAEL
To: MESO SCALE TECHNOLOGIES, LLC.
Reel/Frame 047768/0211 →
Continuity (3)
Continuation 14847761 · Sep 8, 2015
Provisional Application 62047097 · Sep 8, 2014
Related Publication 20180029034A1 · Feb 1, 2018
Cited By (5)
US 12,480,940 US 12,553,887 US 12,631,624 US 12,678,792 US 12,704,459