Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
First Claim
1. A method for detecting the binding of a first member to a second member of a ligand pair, comprising:
- (a) combining a set of first tagged members with a biological sample which may contain one or more second members, under conditions, and for a time sufficient to permit binding of a first member to a second member, wherein said tag is correlative with a particular first member and detectable by non-fluorescent spectrometry or potentiometry;
(b) separating bound first and second members from unbound members;
(c) cleaving said tag from said tagged first member; and
(d) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom detecting the binding of said first member to said second member.
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Accused Products
Abstract
Methods are provided for detecting the binding of a first member to a second member of a ligand pair, comprising the steps of (a) combining a set of first tagged members with a biological sample which may contain one or more second members, under conditions, and for a time sufficient to permit binding of a first member to a second member, wherein said tag is correlative with a particular first member and detectable by non-fluorescent spectrometry, or potentiometry, (b) separating bound first and second members from unbound members, (c) cleaving the tag from the tagged first member, and (d) detecting the tag by non-fluorescent spectrometry, or potentiometry, and therefrom detecting the binding of the first member to the second member.
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Citations
72 Claims
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1. A method for detecting the binding of a first member to a second member of a ligand pair, comprising:
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(a) combining a set of first tagged members with a biological sample which may contain one or more second members, under conditions, and for a time sufficient to permit binding of a first member to a second member, wherein said tag is correlative with a particular first member and detectable by non-fluorescent spectrometry or potentiometry; (b) separating bound first and second members from unbound members; (c) cleaving said tag from said tagged first member; and (d) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom detecting the binding of said first member to said second member. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 56, 57, 58, 59, 60, 61, 72)
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20. A method for analyzing the pattern of gene expression from a selected biological sample, comprising:
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(a) exposing nucleic acids from a biological sample; (b) combining said exposed nucleic acids with one or more selected tagged nucleic acid probes, under conditions and for a time sufficient for said probes to hybridize to said nucleic acids, wherein said tag is correlative with a particular nucleic acid probe and detectable by non-fluorescent spectrometry or potentiomery; (c) separating hybridized probes from unhybridized probes; (d) cleaving said tag from said tagged fragment; and (e) detecting said tag by nonfluorescent spectrometry or potentiometry, and therefrom determining the pattern of gene expression of said biological sample. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 62, 63, 64, 65, 66, 67)
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33. A compound of the formula:
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space="preserve" listing-type="equation">T.sup.ms -L-Xwherein Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a Tms -containing moiety to be cleaved from the remainder of the compound, wherein the Tms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid, the compound having a mass of at least 250 daltons; and-L-X has the formula;
##STR21## wherein one or more of positions b, c, d or e is substituted with hydrogen, alkyl, alkoxy, fluoride, chloride, hydroxyl, carboxylate or amide;
R1 is hydrogen or hydrocarbyl, and R2 comprises a molecule of interest (MOI) other than a nucleic acid fragment. - View Dependent Claims (36, 43, 44, 45, 51)
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34. A compound of the formula:
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space="preserve" listing-type="equation">T.sup.ms -L-Xwherein Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine, and has the formula;
space="preserve" listing-type="equation">T.sup.2 -(J-T.sup.3 -).sub.n -wherein T2 is an organic moiety formed from carbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 15 to 500 daltons;
T3 is an organic moiety formed from carbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 50 to 1000 daltons;
J is a direct bond or a functional group selected from amide, ester, amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine, oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide, sulfonate, sulfonamide or carbon-carbon bond; and
n is an integer ranging from 1 to 50, and when n is greater than 1, each T3 and J is independently selected;L is an organic group which allows a Tms -containing moiety to be cleaved from the remainder of the compound, wherein the Tms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and X comprises a molecules of interest (MOI) other than a nucleic acid fragment, and the compound has a mass of at least 250 daltons. - View Dependent Claims (35, 42, 46, 47, 48, 49, 50, 52)
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37. A compound of the formula:
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space="preserve" listing-type="equation">T.sup.ms -L-Xwherein Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a Tms -containing moiety to be cleaved from the remainder of the compound, wherein the Tms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid;
X comprises a molecule of interest (MOI) other than a nucleic acid fragment, the compound having a mass of at least 250 daltons;
the compound also having the formula;
##STR22## wherein G is (CH2)1-6 wherein a hydrogen on one and only one of the CH, groups is replaced with-(CH2)c -Amide-T4 ;T2 and T4 are organic moieties of the formula C1-25 N0-9 O0-9 H.sub.α
F.sub.β
wherein the sum of α and
β
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;
##STR23## R1 is hydrogen or C1-10 alkyl;
c is an integer ranging from 0 to 4; andn is an integer ranging from 1 to 50 such that when n is greater than 1, then G, c, Amide, R1 and T4 are independently selected. - View Dependent Claims (53)
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38. A compound of the formula:
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space="preserve" listing-type="equation">T.sup.ms -L-Xwherein Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a Tms -containing moiety to be cleaved from the remainder of the compound, wherein the Tms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid;
X comprises a molecule of interest (MOI) other than a nucleic acid fragment, the compound having a mass of at least 250 daltons, the compound also having the formula;
##STR24## wherein G is (CH2)1-6 wherein a hydrogen on one and only one of the CH2 groups is replaced with-(CH2)c -Amide-T4 ;T2 and T4 are organic moieties of the formula C1-25 N0-9 O0-9 H.sub.α
F.sub.β
wherein the sum of α and
β
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;
##STR25## R1 is hydrogen or C1-10 alkyl c is an integer ranging from 0 to 4; andT5 is an organic moiety of the formula C1-25 N0-9 O0-9 H.sub.α
F.sub.β
wherein the sum of α and
β
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms; and
T5 includes a tertiary or quaternary amine or an organic acid; and
m is an integer ranging from 0-49 where when m is greater than 1, then G, c, Amide, R1 and T4 are independently selected. - View Dependent Claims (40, 41, 54)
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39. A compound of the formula:
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space="preserve" listing-type="equation">T.sup.ms -L-Xwherein Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a Tms -containing moiety to be cleaved from the remainder of the compound, wherein the Tms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid;
X comprises a molecule of interest (MOI) other than a nucleic acid fragment, the compound having a mass of at least 250 daltons;
the compound also having the formula;
##STR26## wherein G is (CH2)1-6 wherein a hydrogen on one and only one of the CH2 groups is replaced with-(CH2)c -Amide-T4 ;T2 and T4 are organic moieties of the formula C1-25 N0-9 O0-9 H.sub.α
F.sub.β
wherein the sum of α and
β
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;
##STR27## R1 is hydrogen or C1-10 alkyl;
c is an integer ranging from 0 to 4; andT5 is an organic moiety of the formula C1-25 N0-9 O0-9 H.sub.α
F.sub.β
wherein the sum of α and
β
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms; and
T5 includes a tertiary or quaternary amine or an organic acid; and
m is an integer ranging from 0-49 where when m is greater than 1, then G, c, Amide, R1 and T4 are independently selected. - View Dependent Claims (55)
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68. A composition comprising a pair of compounds of the formula:
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space="preserve" listing-type="equation">T.sup.ms -L-MOIwherein, Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group which allows a Tms -containing moiety to be cleaved from the remainder of the compound, wherein the Tms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; MOI is a nucleic acid fragment wherein L is conjugated to MOI at other than the 3'"'"' end of the MOI; and the compounds of the pair have non-identical Tms groups, and have identical sequences except at one base position where the bases are non-identical. - View Dependent Claims (70, 71)
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69. A composition comprising a pair of compounds of the formula:
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space="preserve" listing-type="equation">T.sup.ms -L-MOIwherein, Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group which allows a Tms -containing moiety to be cleaved from the remainder of the compound, wherein the Tms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; MOI is a nucleic acid fragment wherein L is conjugated to MOI at other than the 3'"'"' end of the MOI; and the compounds of the pair have non-identical Tms groups, and have identical sequences except at two base position where the bases are non-identical.
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Specification