Detection of analytes using fluorescent energy transfer
First Claim
1. A composition of matter, comprising (S1.A)n1 and (S2.D)n2, wherein S1 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, and wherein n1 and n2 represent the stoichiometry of said subunits in said composition.
1 Assignment
0 Petitions
Accused Products
Abstract
The invention provides labeled proteins suitable for determining the presence of cAMP, other second messengers and organic molecules. The proteins are separately labeled with fluorochromes which, when in close spatial proximity, preferably, less than about 6 nm, interact through the transfer of energy from one fluorochrome to the other.
A composition of matter, (S1.A)n1 (S2.D)n2 is provided wherein S1 and S2 are two proteins which are associated in one state and substantially disassociated in another, the equilibrium between which is controlled by the free concentration of an analyte, and A and D are fluorochromes, the emission wavelength of fluorochrome D overlapping the excitation wavelength of fluorochrome A and the distance between A and D being in sufficiently close proximity to allow the radiationless transfer of energy between the fluorochromes. A and D can be selected from a variety of acceptor, donor pairs such as fluorescein and tetramethylrhodamine and derivatives thereof. The concentration of analytes such as cAMP in a sample can be determined by contacting the sample with (S1.A)n1 (S2.D)n2 providing energy near the excitation wavelength of D and measuring the fluorescence of A or D, the concentration of cAMP and other said analytes being determined by the ratio of emission of D to the emission of A, previously calibrated with reference solutions of known analyte concentration.
-
Citations
60 Claims
- 1. A composition of matter, comprising (S1.A)n1 and (S2.D)n2, wherein S1 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, and wherein n1 and n2 represent the stoichiometry of said subunits in said composition.
- 9. A composition of matter, comprising (S1.A)n1 and (S2.D)n2, wherein S1 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and S2 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A and D and A are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, and wherein n1 and n2 represent the stoichiometry of said subunits in said composition.
-
15. A method of determining the concentration of adenosine 3'"'"', 5'"'"'-cyclic monophosphate in a sample comprising:
-
(a) forming the complex represented by the formula (S1.A)n1 and (S2.D)n2, wherein S1 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, and wherein n1 and n2 represent the stoichiometry of said subunits in said complex; (b) contacting the complex with the sample; (c) exposing said sample to radiative energy near the excitation wavelength of D; and (d) measuring the fluorescence emitted at the emission wavelength of at least one of D and A is. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
-
-
28. A method of determining the concentration of adenosine 3'"'"', 5'"'"'-cyclic monophosphate in a sample comprising:
-
(a) forming a complex represented by the formula (S1.A)n1 and (S2.D)n2, wherein S1 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, and wherein n1 and n2 represent the stoichiometry of said subunits in said complex; (b) contacting the complex with the sample; (c) exposing said sample to radiative energy near the excitation wavelength of D and (d) measuring the fluorescence emitted at the emission wavelength of at least one of D and A. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
-
-
41. A method of preparing a labeled complex comprising (S1.A) and (S2.D), wherein S1 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, said method comprising:
-
(a) mixing the labeled subunits S1 .A and S2.D under conditions suitable for the formation of a complex comprising {(S1.A)n1.(S2 .D)n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits in said complex; and
wherein said fluorochromes are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes; and(b) recovering the complex. - View Dependent Claims (42, 43, 44, 45)
-
-
46. A method of preparing a labeled complex comprising (S1.A) and (S2.D), wherein S1 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, wherein a first complex comprising S1 and S2 is attached to fluorochrome A, and a second complex comprising S1 and S2 is attached to fluorochrome D;
- wherein the subunits of said first and second complexes are separated to obtain labeled subunits S1.D, S2.D, S1.A and S2.A;
said method comprising mixing said labeled subunits under conditions such that said fluorochromes come in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes to occur, resulting in the formation of labeled complexes {(S1.D)n1.(S2.A)n2 } or {(S1.A)n1.(S2.D)n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits. - View Dependent Claims (47, 48, 49, 50)
- wherein the subunits of said first and second complexes are separated to obtain labeled subunits S1.D, S2.D, S1.A and S2.A;
-
51. A method of preparing a labeled complex comprising (S1.A) and (S2.D), wherein S1 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, said method comprising:
-
(a) mixing the labeled subunits S1.A and S2.D under conditions suitable for the formation of a complex comprising {(S1.A)n1.(S2.D)n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits in said complex; and
wherein said fluorochromes are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes; and(b) recovering the complex. - View Dependent Claims (52, 53, 54, 55)
-
-
56. A method of preparing a labeled complex comprising (S1 .A) and (S2.D), wherein S1 is the catalytic subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase and S2 is the regulatory subunit of adenosine 3'"'"', 5'"'"'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlaps the excitation wavelength of the other A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, wherein a first complex comprising S1 and S2 is attached to fluorochrome A, and a second complex comprising S1 and S2 is attached to fluorochrome D;
- wherein the subunits of said first and second complexes are separated to obtain labeled subunits S1.D, S2.D, S1.A and S2.A;
said method comprising mixing said labeled subunits under conditions such that said fluorochromes come in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes to occur, resulting in the formation of labeled complexes {(S1.D)n1.(S2.A)n2 } or {(S1.A)n1.(S2.D)n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits. - View Dependent Claims (57, 58, 59, 60)
- wherein the subunits of said first and second complexes are separated to obtain labeled subunits S1.D, S2.D, S1.A and S2.A;
Specification