US5439797A
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US
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Detection of analytes using fluorescent energy transfer
Assignee: University of California, San Diego
•
Filing: 1993-08-30
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Granted: 1995-08-08
•
Expires: 2013-08-30
Abstract
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, (S 1 .A) n1 (S 2 .D) n2 is provided wherein S 1 and S 2 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 (S 1 .A) n1 (S 2 .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.
Inventors
- 👤 Roger Y. Tsien
- 👤 Susan S. Taylor
- 👤 Stephen R. Adams
- 👤 Ying Ji
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 13:35
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/542; G01N33/58; G01N33/582; Y; Y10; Y10S; Y10S436/00; Y10S436/80
Claims Analysis
Total Claims: 68
Claim 1
Independent Claim
96 words
A composition of matter, comprising (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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.
Claim 2
Dependent Claim
96 words
A composition of matter, comprising (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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.
Claim 3
Dependent Claim
47 words
The composition of matter of claim 1, wherein D and A are selected from the group consisting of: fluorescein and tetramethylrhodamine, a 7-aminocoumarin and dipyrromethene-borate, an indocarbocyanine and rhodamine X, and a phycoerythrin and allophycocyanin, or derivatives thereof, provided that the selected pair exhibit fluorescence energy transfer.
Claim 4
Dependent Claim
25 words
The composition of matter of claim 1, wherein the emission wavelength of D and the excitation wavelength of A are about 510 to 550 nm.
Claim 5
Dependent Claim
17 words
The composition of matter of claim 3, wherein the fluorochromes are fluorescein and tetramethylrhodamine or derivatives thereof.
Claim 6
Dependent Claim
15 words
The composition of matter of claim 1, wherein n1 is 2 and n2 is 2.
Claim 7
Dependent Claim
16 words
The composition of matter of claim 1, wherein S 1 and S 2 are recombinant proteins.
Claim 8
Dependent Claim
22 words
The composition of matter of claim 1, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 9
Dependent Claim
18 words
The composition of matter of claim 1, wherein S 1 and S 2 are derived from mammalian sources.
Claim 10
Dependent Claim
96 words
A composition of matter, comprising (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and S 2 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.
Claim 11
Dependent Claim
96 words
A composition of matter, comprising (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and S 2 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.
Claim 12
Dependent Claim
45 words
The composition of claim 9, wherein D and A are selected from the group consisting of: fluorescein and tetramethylrhodamine; a 7-aminocoumarin and dipyrromethene-borate; an indocarbocyanine and rhodamine X; and a phycoerythrin and allophycocyanin, or derivatives thereof, provided that the selected pair exhibit fluorescence energy transfer.
Claim 13
Dependent Claim
25 words
The composition of matter of claim 9, wherein the emission wavelength of D and the excitation wavelength of A are about 510 to 550 nm.
Claim 14
Dependent Claim
16 words
The composition of matter of claim 9, wherein S 1 and S 2 are recombinant proteins.
Claim 15
Dependent Claim
22 words
The composition of matter of claim 9, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 16
Dependent Claim
18 words
The composition of matter of claim 9, wherein S 1 and S 2 are derived from mammalian sources.
Claim 17
Independent Claim
156 words
A method of determining the concentration of adenosine 3', 5'-cyclic monophosphate in a sample comprising: (a) forming the complex represented by the formula (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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.
Claim 18
Independent Claim
156 words
A method of determining the concentration of adenosine 3', 5'-cyclic monophosphate in a sample comprising: (a) forming the complex represented by the formula (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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.
Claim 19
Dependent Claim
50 words
The method of claim 15, wherein the measuring step comprises measuring the fluorescence emitted at the emission wavelength of both A and D, wherein the extent of energy transfer from D to A is determined by the ratio of the emission amplitude of A to the emission amplitude of D.
Claim 20
Dependent Claim
45 words
The method of claim 15, wherein the measuring step comprises measuring the fluorescence emitted at the emission wavelength of D and determining the extent of the energy transfer from D to A by the fluorescence lifetime of D or the rate of photobleaching of D.
Claim 21
Dependent Claim
23 words
The method of claim 15, wherein the sample comprises an intact cell and the contacting step comprises incorporating the complex into the cell.
Claim 22
Dependent Claim
24 words
The method of claim 18, wherein the contacting step is selected from the group consisting of microinjection, electroporation, lipofection, bead loading and scrape loading.
Claim 23
Dependent Claim
45 words
The method of claim 15, wherein D and A are selected from the group consisting of: fluorescein and tetramethylrhodamine; a 7-aminocoumarin and dipyrromethene-borate; an indocarbocyanine and rhodamine X; and a phycoerythrin and allophycocyanin, or derivatives thereof, provided that the selected pair exhibit fluorescence energy transfer.
Claim 24
Dependent Claim
23 words
The method of claim 15, wherein the emission wavelength of D and the excitation wavelength of A are about 510 to 550 nm.
Claim 25
Dependent Claim
17 words
The method of claim 20 or 21, wherein the fluorochromes are fluorescein and tetramethylrhodamine or derivatives thereof.
Claim 26
Dependent Claim
13 words
The method of claim 15, wherein n1 is 2 and n2 is 2.
Claim 27
Dependent Claim
14 words
The method of claim 15, wherein S 1 and S 2 are recombinant proteins.
Claim 28
Dependent Claim
20 words
The method of claim 15, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 29
Dependent Claim
16 words
The method of claim 15, wherein S 1 and S 2 are derived from mammalian sources.
Claim 30
Dependent Claim
39 words
A method according to claim 15, wherein the measuring step comprises measuring the fluorescence emitted at the emission wavelength of A, wherein the extent of energy transfer from D to A is determined by the amplitude of said emission.
Claim 31
Independent Claim
155 words
A method of determining the concentration of adenosine 3', 5'-cyclic monophosphate in a sample comprising: (a) forming a complex represented by the formula (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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.
Claim 32
Independent Claim
155 words
A method of determining the concentration of adenosine 3', 5'-cyclic monophosphate in a sample comprising: (a) forming a complex represented by the formula (S 1 .A) n1 and (S 2 .D) n2 , wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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.
Claim 33
Dependent Claim
50 words
The method of claim 28, wherein the measuring step comprises measuring the fluorescence emitted at the emission wavelengths of both A and D, wherein the extent of energy transfer from D to A is determined by the ratio of the emission amplitude of A to the emission amplitude of D.
Claim 34
Dependent Claim
45 words
The method of claim 28, wherein the measuring step comprises measuring the fluorescence emitted at the emission wavelength of D and determining the extent of the energy transfer from D to A by the fluorescence lifetime of D or the rate of photobleaching of D.
Claim 35
Dependent Claim
23 words
The method of claim 28, wherein the sample comprises an intact cell and the contacting step comprises incorporating the complex into the cell.
Claim 36
Dependent Claim
24 words
The method of claim 31, wherein the contacting step is selected from the group consisting of microinjection, electroporation, lipofection, bead loading and scrape loading.
Claim 37
Dependent Claim
45 words
The method of claim 28, wherein D and A are selected from the group consisting of: fluorescein and tetramethylrhodamine; a 7-aminocoumarin and dipyrromethene-borate; an indocarbocyanine and rhodamine X; and a phycoerythrin and allophycocyanin, or derivatives thereof, provided that the selected pair exhibit fluorescence energy transfer.
Claim 38
Dependent Claim
23 words
The method of claim 28, wherein the emission wavelength of D and the excitation wavelength of A are about 510 to 550 nm.
Claim 39
Dependent Claim
17 words
The method of claim 33 or 34, wherein the fluorochromes are fluorescein and tetramethylrhodamine or derivatives thereof.
Claim 40
Dependent Claim
13 words
The method of claim 28, wherein n1 is 2 and n2 is 2.
Claim 41
Dependent Claim
14 words
The method of claim 28, wherein S 1 and S 2 are recombinant proteins.
Claim 42
Dependent Claim
20 words
The method of claim 28, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 43
Dependent Claim
16 words
The method of claim 28, wherein S 1 and S 2 are derived from mammalian sources.
Claim 44
Dependent Claim
39 words
A method according to claim 28, wherein the measuring step comprises measuring the fluorescence emitted at the emission wavelength of A, wherein the extent of energy transfer from D to A is determined by the amplitude of said emission.
Claim 45
Independent Claim
153 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 .A and S 2 .D under conditions suitable for the formation of a complex comprising {(S 1 .A) n1 .(S 2 .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.
Claim 46
Independent Claim
153 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 .A and S 2 .D under conditions suitable for the formation of a complex comprising {(S 1 .A) n1 .(S 2 .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.
Claim 47
Dependent Claim
15 words
The method of claim 41, wherein the fluorochromes are fluorescein and tetramethylrhodamine, or derivatives thereof.
Claim 48
Dependent Claim
14 words
The method of claim 41, wherein S 1 and S 2 are recombinant proteins.
Claim 49
Dependent Claim
20 words
The method of claim 41, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 50
Dependent Claim
16 words
The method of claim 41, wherein S 1 and S 2 are derived from mammalian sources.
Claim 51
Independent Claim
209 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 and S 2 is attached to fluorochrome A, and a second complex comprising S 1 and S 2 is attached to fluorochrome D; wherein the subunits of said first and second complexes are separated to obtain labeled subunits S 1 .D, S 2 .D, S 1 .A and S 2 .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 {(S 1 .D) n1 .(S 2 .A) n2 } or {(S 1 .A) n1 .(S 2 .D) n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits.
Claim 52
Independent Claim
209 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 and S 2 is attached to fluorochrome A, and a second complex comprising S 1 and S 2 is attached to fluorochrome D; wherein the subunits of said first and second complexes are separated to obtain labeled subunits S 1 .D, S 2 .D, S 1 .A and S 2 .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 {(S 1 .D) n1 .(S 2 .A) n2 } or {(S 1 .A) n1 .(S 2 .D) n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits.
Claim 53
Dependent Claim
15 words
The method of claim 46, wherein the fluorochromes are fluorescein and tetramethylrhodamine, or derivatives thereof.
Claim 54
Dependent Claim
14 words
The method of claim 46, wherein S 1 and S 2 are recombinant proteins.
Claim 55
Dependent Claim
20 words
The method of claim 46, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 56
Dependent Claim
16 words
The method of claim 46, wherein S 1 and S 2 are derived from mammalian sources.
Claim 57
Independent Claim
156 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 .A and S 2 .D under conditions suitable for the formation of a complex comprising {(S 1 .A) n1 .(S 2 .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.
Claim 58
Independent Claim
156 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 .A and S 2 .D under conditions suitable for the formation of a complex comprising {(S 1 .A) n1 .(S 2 .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.
Claim 59
Dependent Claim
15 words
The method of claim 51, wherein the fluorochromes are fluorescein and tetramethylrhodamine, or derivatives thereof.
Claim 60
Dependent Claim
14 words
The method of claim 51, wherein S 1 and S 2 are recombinant proteins.
Claim 61
Dependent Claim
20 words
The method of claim 51, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 62
Dependent Claim
16 words
The method of claim 51, wherein S 1 and S 2 are derived from mammalian sources.
Claim 63
Independent Claim
211 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 and S 2 is attached to fluorochrome A, and a second complex comprising S 1 and S 2 is attached to fluorochrome D; wherein the subunits of said first and second complexes are separated to obtain labeled subunits S 1 .D, S 2 .D, S 1 .A and S 2 .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 {(S 1 .D) n1 .(S 2 .A) n2 } or {(S 1 .A) n1 .(S 2 .D) n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits.
Claim 64
Independent Claim
211 words
A method of preparing a labeled complex comprising (S 1 .A) and (S 2 .D), wherein S 1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S 2 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 S 1 and S 2 is attached to fluorochrome A, and a second complex comprising S 1 and S 2 is attached to fluorochrome D; wherein the subunits of said first and second complexes are separated to obtain labeled subunits S 1 .D, S 2 .D, S 1 .A and S 2 .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 {(S 1 .D) n1 .(S 2 .A) n2 } or {(S 1 .A) n1 .(S 2 .D) n2 }, wherein n1 and n2 represent the stoichiometry of the labeled subunits.
Claim 65
Dependent Claim
15 words
The method of claim 56, wherein the fluorochromes are fluorescein and tetramethylrhodamine, or derivatives thereof.
Claim 66
Dependent Claim
14 words
The method of claim 56, wherein S 1 and S 2 are recombinant proteins.
Claim 67
Dependent Claim
20 words
The method of claim 56, wherein one protein is a recombinant protein and one protein is derived from mammalian sources.
Claim 68
Dependent Claim
16 words
The method of claim 56, wherein S 1 and S 2 are derived from mammalian sources.
References Cited (Backward Citations)
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