Sensitivity in fluorescence assays in icteric samples
First Claim
1. In an enzyme immunoassay method for the determination of an analyte in an icteric blood sample suspected of containing said analyte, said method comprising combining in an assay medium said sample and assay reagents and irradiating said medium, said method involving the production or oxidation of NADH or NADPH in said medium and employing fluorescent detection of NADH or NADPH wherein a change in fluorescence is related to the amount of analyte in said sample, the improvement which comprises irradiating said medium containing said sample and said assay reagents with light below 400 nm to excite NADH or NADPH produced or oxidized in said assay medium and detecting the fluorescence of NADH or NADPH at a wavelength at or above 500 nm.
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Abstract
Enhanced sensitivity in fluorescent assays employing NADH or NADPH is obtained, particularly in icteric samples, by employing excitation light at a wave length range below about 400 nm and reading emitted light above about 500 nm.
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Citations
10 Claims
- 1. In an enzyme immunoassay method for the determination of an analyte in an icteric blood sample suspected of containing said analyte, said method comprising combining in an assay medium said sample and assay reagents and irradiating said medium, said method involving the production or oxidation of NADH or NADPH in said medium and employing fluorescent detection of NADH or NADPH wherein a change in fluorescence is related to the amount of analyte in said sample, the improvement which comprises irradiating said medium containing said sample and said assay reagents with light below 400 nm to excite NADH or NADPH produced or oxidized in said assay medium and detecting the fluorescence of NADH or NADPH at a wavelength at or above 500 nm.
- 6. In an enzyme immunoassay method for the determination of an analyte in an icteric blood sample suspected of containing said analyte, said method comprising combining in an assay medium said sample and assay reagents and irradiating said medium, said method involving employing in said assay medium an enzyme, a substrate for said enzyme, and employing NAD or NADP as a cofactor for said enzyme, said method involving production of the reduced form of NAD or NADP in said medium and employing detection of the reduced form of NAD or NADP in said medium by measuring the light emitted by said reduced form of NAD or NADP, the improvement which comprises irradiating said assay medium containing said sample and said assay reagents suspected of containing said reduced form of NAD or NADP with light below about 400 nm and measuring the light emitted by said reduced form at a wavelength at or above 500 nm.
Specification