Homogeneous specific binding assay employing an intramolecularly modulated photogenic enzyme substrate label
First Claim
1. In a homogeneous specific binding assay method for determining a ligand in, or the ligand binding capacity of, a liquid medium,wherein a liquid reaction mixture is obtained by combining said liquid medium with reagent means including(1) a labeled conjugate having a label component and a binding component, which conjugate is cleavable by an enzyme to produce an indicator product which emits light upon exposure to excitation means, and(2) said enzyme which cleaves said labeled conjugate,thereby forming in said reaction mixture a binding reaction system having a bound-species and a free-species of said labeled conjugate, the activity of said labeled conjugate as a substrate for said cleaving enzyme being substantially different in said bound-species compared to said free-species, andwherein said reaction mixture is exposed to said excitation means and resulting light emitted is measured as a function of the amount of said ligand in, or said ligand binding capacity of, said liquid medium,the improvement which comprises employing as said label component of said labeled conjugate, a residue having the formula:
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space="preserve" listing-type="equation">P--X--M--R wherein P is a photophore which emits light upon exposure to said excitation means, X is a linkage which is cleavable by said enzyme, M is a modulator for said light emission of said photophore, and R is a linking group through which said label component is covalently bound to the binding component in said conjugate, said labeled conjugate and said cleaved indicator product emitting substantially different amounts of light upon exposure to said excitation means due to modulation of the emission of P by the proximity of M in said labeled conjugate.
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Abstract
A homogeneous specific binding assay method and reagent means for determining a ligand, such as a hapten, antigen or antibody, in, or the ligand binding capacity of, a liquid medium employing a labeled conjugate which upon enzymatic cleavage produces a detectable indicator product. The improvement comprises employing as the label component in the labeled conjugate, a residue having the formula:
P--X--M--R
wherein P is a photophore, e.g., a fluorescer, which emits light upon exposure to excitation means, X is an enzymatically cleavable linkage, M is a modulator, e.g., a quencher, for said light emission of the photophore, and R is a linking group to the binding component, e.g., the ligand or analog thereof, in the labeled conjugate. The labeled conjugate and the cleaved indicator product emit substantially different amounts of light upon excitation due to modulation of the emission of photophore P by the proximity of modulator M in the labeled conjugate.
41 Citations
40 Claims
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1. In a homogeneous specific binding assay method for determining a ligand in, or the ligand binding capacity of, a liquid medium,
wherein a liquid reaction mixture is obtained by combining said liquid medium with reagent means including (1) a labeled conjugate having a label component and a binding component, which conjugate is cleavable by an enzyme to produce an indicator product which emits light upon exposure to excitation means, and (2) said enzyme which cleaves said labeled conjugate, thereby forming in said reaction mixture a binding reaction system having a bound-species and a free-species of said labeled conjugate, the activity of said labeled conjugate as a substrate for said cleaving enzyme being substantially different in said bound-species compared to said free-species, and wherein said reaction mixture is exposed to said excitation means and resulting light emitted is measured as a function of the amount of said ligand in, or said ligand binding capacity of, said liquid medium, the improvement which comprises employing as said label component of said labeled conjugate, a residue having the formula: -
space="preserve" listing-type="equation">P--X--M--Rwherein P is a photophore which emits light upon exposure to said excitation means, X is a linkage which is cleavable by said enzyme, M is a modulator for said light emission of said photophore, and R is a linking group through which said label component is covalently bound to the binding component in said conjugate, said labeled conjugate and said cleaved indicator product emitting substantially different amounts of light upon exposure to said excitation means due to modulation of the emission of P by the proximity of M in said labeled conjugate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. In a homogeneous specific binding assay method for determining a ligand in a liquid medium, or for determining the ligand binding capacity of a liquid medium suspected to contain a specific binding partner of said ligand,
wherein a liquid reaction mixture is obtained by combining said liquid medium with (1) a labeled conjugate having a label component coupled to said ligand or a binding analog thereof, which conjugate is cleavable by an enzyme to produce an indicator product which fluoresces upon excitation with light of a predetermined first wavelength, emitting light at a second wavelength, (2) said enzyme which cleaves said labeled conjugate, and, (3) where said ligand is under determination, a specific binding partner of said ligand, and the activity of said labeled conjugate as a substrate for said cleaving enzyme being substantially decreased by binding of said specific binding partner, and wherein said reaction mixture is irradiated with light of said predetermined first wavelength and resulting fluorescence is measured as a function of the amount of said ligand in, or said ligand binding capacity of, said liquid medium, the improvement which comprises employing as said label component of said labeled conjugate, a residue having the formula: -
space="preserve" listing-type="equation">F--X--Q--Rwherein F is a fluorescer which is excited by light of said predetermined first wavelength to emit light at said second wavelength, X is a linkage which is cleavable by said enzyme, Q is a quencher which when proximal to fluorescer F absorbs the fluorescent emission of fluorescer F at said second wavelength, and R is a linking group through which said label component is covalently bound to said ligand or analog thereof in said conjugate, said labeled conjugate exhibiting substantially less fluorescence at said second wavelength than said cleaved indicator product upon irradiation with light of said predetermined first wavelength due to quenching of the fluorescence of F by proximity of Q in said labeled conjugate. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22)
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23. In a reagent means for use in a homogeneous specific binding assay method for determining a ligand in, or the ligand binding capacity of, a liquid medium, which means comprises
(1) a labeled conjugate having a label component and a binding component, which conjugate is cleavable by an enzyme to produce an indicator product which emits light upon exposure to excitation means, and (2) said enzyme which cleaves said labeled conjugate, the improvement wherein said label component of said labeled conjugate is a residue having the formula: -
space="preserve" listing-type="equation">P--X--M--Rwherein P is a photophore which emits light upon exposure to such excitation means, X is a linkage which is cleavable by said enzyme, M is a modulator for said light emission of said photophore, and R is a linking group through which said label component is covalently bound to the binding component in said conjugate, said labeled conjugate and said cleaved indicator product emitting substantially different amounts of light upon exposure to said excitation means due to modulation of the emission of P by the proximity of M in said labeled conjugate. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. In a reagent means for use in a homogeneous specific binding assay method for determining a ligand in a liquid medium, or for determining the ligand binding capacity of a liquid medium suspected to contain a specific binding partner of said ligand, which means comprises
(1) a labeled conjugate having a label component coupled to said ligand or a binding analog thereof, which conjugate is cleavable by an enzyme to produce an indicator product which fluoresces upon excitation with light of a predetermined first wavelength, emitting light at a second wavelength, (2) said enzyme which cleaves said labeled conjugate, and (3) where said ligand is under determination, a specific binding partner of said ligand, the improvement which comprises employing as said label component of said labeled conjugate, a residue having the formula: -
space="preserve" listing-type="equation">F--X--Q--Rwherein F is a fluorescer which is excited by light of said predetermined first wavelength to emit light at said second wavelength, X is a linkage which is cleavable by said enzyme, Q is a quencher which when proximal to fluorescer F absorbs the fluorescent emission of fluorescer F at said second wavelength, and R is a linking group through which said label component is covalently bound to said ligand or analog thereof in said conjugate, said labeled conjugate exhibiting substantially less fluorescence at said second wavelength than said cleaved indicator product upon irradiation with light of said predetermined first wavelength due to quenching of the fluorescence of F by proximity of Q in said labeled conjugate. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40)
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Specification