Methods for conducting multiplexed assays
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.
1 . A method of conducting a multiplexed binding assay for a plurality of analytes comprising a first analyte of interest 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 which is complementary to the oligonucleotide of the first targeting agent and forms 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 of interest,
(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 which is complementary to the oligonucleotide of the second targeting agent and forms a second oligonucleotide pair, and
(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 of interest, wherein,
the first linking agent binds to the first supplemental linking agent and the second linking agent binds to 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 of interest, 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 of interest, and
(c) measuring the amount of the first analyte of interest and second analytes analyte of interest on the first binding domain and the second binding domain, respectively.
2 . The method of claim 1 , wherein the sample further comprises one or more additional analytes of interest comprising a first additional analyte of interest, and step (a) further comprises
combining, in one or more steps, the components of step (a) with an additional targeting agent immobilized on an additional binding domain, an additional targeting agent complement connected to an additional linking agent, wherein the additional targeting agent complement is a binding partner of the additional targeting agent, and an additional binding reagent connected to an additional supplemental linking agent, wherein the additional binding reagent is a binding partner of the first additional analyte of interest, and wherein the additional linking agent binds to the additional supplemental linking agent;
step (b) further comprises forming an additional binding complex on the additional binding domain comprising the additional targeting agent, the additional targeting agent complement, the additional binding reagent and the first additional analyte of interest; and
step (c) further comprises measuring the amount of the first additional analyte of interest on the additional binding domain.
3 . The method of claim 1 , wherein step (a) further comprises the steps of:
combining, in a first volume of liquid, the first targeting agent complement and the first binding reagent and forming a first targeting complex by linking the first targeting agent complement to the first binding reagent through the first linking agent and the first supplemental linking agent; and
combining, in a second volume of liquid, the second targeting agent complement and the second binding reagent and forming a second targeting complex by linking the second targeting agent complement to the second binding reagent through the second linking agent and the second supplemental linking agent.
4 . The method of claim 3 , wherein step (a) further comprises the steps of:
forming a combination of the first targeting complex and the second targeting complex by combining the first targeting complex and the second targeting complex,
contacting the combination of the first targeting complex and the second targeting complex with the first binding domain and the second binding domain, and
binding the first targeting complex to the first targeting agent on the first binding domain and binding the second targeting complex to the second targeting agent on the second binding domain.
5 . The method of claim 4 , wherein the first targeting complex and the second targeting complex are combined with the sample prior to contacting the first targeting complex and the second targeting complex with the first binding domain and the second binding domain.
6 . The method of claim 5 , wherein the first analyte of interest binds to the first binding reagent in the first targeting complex and the second analyte of interest binds to the second binding reagent in the second targeting complex.
7 . The method of claim 4 , wherein the first targeting complex and the second targeting complex are combined with the sample after contacting the first targeting complex and the second targeting complex with the first binding domain and the second binding domain.
8 . The method of claim 7 , wherein the first analyte of interest binds to the first binding reagent in the first targeting complex and the second analyte of interest binds to the second binding reagent in the second targeting complex.
9 . The method of claim 4 , wherein the first targeting complex and the second targeting complex are combined with the sample and contacted with the first binding domain and the second binding domain at the same time.
10 . The method of claim 9 , wherein the first analyte of interest binds to the first binding reagent in the first targeting complex and the second analyte of interest binds to the second binding reagent in the second targeting complex.
11 . The method of claim 3 , wherein step (a) further comprises:
producing a mixture by combining the first targeting complex and the second targeting complex with the sample,
wherein, in the mixture, the first analyte of interest binds to the first binding reagent in the first targeting complex and the second analyte of interest binds to the second binding reagent in the second targeting complex, and
contacting the mixture of the first targeting complex and the second targeting complex, respectively, with the first binding domain and the second binding domain,
wherein the first targeting complex binds to the first targeting agent on the first binding domain and the second targeting complex binds to the second targeting agent on the second binding domain.
12 . The method of claim 1 , wherein the first linking agent is bound to the first supplemental linking agent and the second linking agent is bound to the second supplemental linking agent through binding interactions between (a) a thiol group and a maleimide or iodoacetamide groups; (b) an aldehyde and a hydrazide; or (c) an alkyne and an azide.
13 . The method of claim 1 , wherein (a) each of the first linking agent and the second linking agent is biotin and each of the first supplemental linking agent and the second supplemental linking agent is streptavidin or avidin; or (b) each of the first linking agent and the second linking agent is streptavidin or avidin and each of the first supplemental linking agent and the second supplemental linking agent is biotin.
14 . The method of claim 1 , wherein the first binding reagent and the second binding reagent are antigens.
15 . The method of claim 1 , wherein the first binding reagent and the second binding reagent are antibodies.
16 . The method of claim 1 , wherein the method is a sandwich binding assay.
17 . 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 and a second detection reagent that binds the second analyte of interest, and wherein the first binding complex further comprises the first detection reagent and the second binding complex further comprises the second detection reagent.
18 . The method of claim 17 , wherein the first detection reagent comprises a first detectable label, wherein the second detection reagent comprises a second detectable label and wherein each of the first detectable label and the second detectable label is an electrochemiluminescent label and the measuring step (c) further comprises applying a voltage waveform to an electrode and measuring electrochemiluminescent signals wherein the first binding domain and the second binding domain are positioned on the electrode, and wherein an amount of an electrochemiluminescent signal from the first detectable label on the first binding complex correlates with an amount of first analyte in the sample and an amount of an electrochemiluminescent signal from the second detectable label on the second binding complex correlates with an amount of second analyte in the sample.
19 . The method of claim 1 , wherein the method is a competitive binding assay wherein the components of step (a) further comprise a first detection reagent that competes with the first analyte of interest for binding to the first binding reagent and a second detection reagent that competes with the second analyte of interest for binding to the second binding reagent.
20 . The method of claim 1 , wherein each of the first binding domain and the second binding domain is an element of an array of binding domains.
21 . The method of claim 1 , wherein each of the first binding domain and the second binding domain is positioned on a surface of a microparticle.
22 . The method of claim 1 , wherein the first oligonucleotide pair and the second oligonucleotide pair are different and each of the first oligonucleotide pair and the second oligonucleotide pair is 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)
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)
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)
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)
23
ttccacttaggg (SEQ ID NO: 45)
ccctaagtggaa (SEQ ID NO: 46)
25
tttcccttgcta (SEQ ID NO: 49)
tagcaagggaaa (SEQ ID NO: 50).
23 . The method of claim 1 , wherein the first binding reagent binds to the first analyte of interest and the second binding reagent binds the second analyte of interest.
24 . The method of claim 23 , wherein the first analyte of interest and the second analyte of interest are different analytes.
25 . The method of claim 1 , wherein the first targeting agent and the second targeting agent and the first targeting agent complement and the second targeting agent complement, respectively, are used to map the binding regions of the first binding reagent and the second binding reagent of a set of binding reagents to the first binding domain and the second binding domain of a set of binding domains and each binding reagent in the set of binding reagents binds to a different analyte of interest of the plurality of analytes in the sample.
26 . The method of claim 1 , wherein the plurality of analytes only has two analytes.