Method of determination of fluorescent substance and method of assay of enzyme activity
First Claim
1. A method for determining the concentration of a fluorescent substance, comprising adding a known amount of a reference fluorescent substance to a specimen containing an objective fluorescent substance to be determined;
- irradiating the specimen with excitation light capable of exciting both the objective fluorescent substance and the reference fluorescent substance;
measuring a first fluorescence intensity at a first wavelength where fluorescence is emitted mainly by the objective fluorescent substance;
measuring a second fluorescence intensity at a second wavelength where fluorescence is emitted mainly by the reference fluorescent substance; and
calculating the concentration of the objective fluorescent substance from the equation;
space="preserve" listing-type="equation">1/(y-c)=a/x+bwhere x is the concentration of the objective fluorescent substance, y is a ratio of the first fluorescence intensity to the second fluorescence intensity, and a, b and c are constants determined by measuring fluorescence intensities of reference specimens containing at least three different concentrations of the objective fluorescent substance.
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Abstract
A method for determining a fluorescent substance in a broader concentration range with higher measurement accuracy is provided which is applicable to assay of enzyme activity in enzyme-label immunoassay, enzyme-label DNA hybridization, etc. A method for assaying enzyme activity is also provided which employs the above fluorescence measurement. In the method, a specimen containing an objective fluorescent substance to be determined and a reference substance is irradiated with excitation light capable of exciting both the objective fluorescent substance and the reference fluorescent substance; measuring a first fluorescence intensity at a first wavelength where fluorescence is emitted mainly by the objective fluorescent substance; measuring a second fluorescence intensity at a second wavelength where fluorescence is emitted mainly by the reference fluorescent substance; and calculating the concentration of the objective fluorescent substance from the equation:
1/(y-c)=a/x+b
where x is the concentration of the objective fluorescent substance, and y is a ratio of the first fluorescence intensity to the second fluorescence intensity.
5 Citations
5 Claims
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1. A method for determining the concentration of a fluorescent substance, comprising adding a known amount of a reference fluorescent substance to a specimen containing an objective fluorescent substance to be determined;
- irradiating the specimen with excitation light capable of exciting both the objective fluorescent substance and the reference fluorescent substance;
measuring a first fluorescence intensity at a first wavelength where fluorescence is emitted mainly by the objective fluorescent substance;
measuring a second fluorescence intensity at a second wavelength where fluorescence is emitted mainly by the reference fluorescent substance; and
calculating the concentration of the objective fluorescent substance from the equation;
space="preserve" listing-type="equation">1/(y-c)=a/x+bwhere x is the concentration of the objective fluorescent substance, y is a ratio of the first fluorescence intensity to the second fluorescence intensity, and a, b and c are constants determined by measuring fluorescence intensities of reference specimens containing at least three different concentrations of the objective fluorescent substance. - View Dependent Claims (3, 4, 5)
- irradiating the specimen with excitation light capable of exciting both the objective fluorescent substance and the reference fluorescent substance;
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2. A method for assaying activity of an enzyme by measuring change with time of concentration of an objective fluorescent substance by action of the enzyme, comprising adding a known concentration of a reference fluorescent substance to a specimen containing an objective fluorescent substance to be determined;
- irradiating the specimen with excitation light capable of exciting both the objective fluorescent substance and the reference fluorescent substance;
measuring a first fluorescence intensity at a first wavelength where fluorescence is emitted mainly by the objective fluorescent substance;
measuring a second fluorescence intensity at a second wavelength where fluorescence is emitted mainly by the reference fluorescent substance;
calculating the concentration of the objective fluorescent substance from the equation;
space="preserve" listing-type="equation">1/(y-c)=a/x+bwhere x is the concentration of the objective fluorescent substance, y is a ratio of the first fluorescence intensity to the second fluorescence intensity, and a, b and c are constants determined by measuring fluorescence intensities of reference specimens containing at least three different concentrations of the objective fluorescent substance; and
deriving the activity of the enzyme or the concentration of the enzyme from the change with time of the value of x.
- irradiating the specimen with excitation light capable of exciting both the objective fluorescent substance and the reference fluorescent substance;
Specification