QUANTITATIVE BINDING ASSAYS USING GREEN FLUORESCENT PROTEIN AS A LABEL
First Claim
1. A method of determining an unknown amount of an analyte in a fluid sample comprising:
- (e) contacting the unknown amount of analyte with a solution having a predefined ratio of a ligand-GFP conjugate, solution volume, and anti-ligand, said anti-ligand being immobilized on a support and having a specific binding affinity for the ligand-GFP conjugate and the analyte;
(f) incubating the analyte with said solution for a predetermined time;
(g) separating the supernatant of said solution from the support;
(h) measuring the intensity of fluorescence of the supernatant; and
(i) relating the measured intensity of fluorescence to the amount of analyte in the sample.
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Abstract
A heterogeneous binding assay for an analyte in a fluid sample is developed, which uses a green fluorescent protein (GFP) label. A ligand-GFP conjugate has a specific binding affinity for an anti-ligand immobilized on a support. The anti-ligand also has a specific binding affinity for the analyte. Competition between the analyte and ligand-GFP conjugate for binding sites on the anti-ligand permits an assay for an unknown amount of the analyte. Preferred specific binding pairs for use in the assay are biotin:avidin, and a selected antibody and its antigen. A preferred assay employing an antibody and its antigen is illustrated for a fusion protein containing GFP and an antigenic determinant. Picomolar amounts of analyte can be detected. The mutant of GFP that contains a six-histidine tail to facilitate purification on an immobilized metal affinity column is chemically modified to incorporate biotin moieties. The resulting conjugates retain the fluorescence characteristics of the unmodified protein and are used along with avidin-coated magnetic beads in the development of the assay.
15 Citations
14 Claims
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1. A method of determining an unknown amount of an analyte in a fluid sample comprising:
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(e) contacting the unknown amount of analyte with a solution having a predefined ratio of a ligand-GFP conjugate, solution volume, and anti-ligand, said anti-ligand being immobilized on a support and having a specific binding affinity for the ligand-GFP conjugate and the analyte;
(f) incubating the analyte with said solution for a predetermined time;
(g) separating the supernatant of said solution from the support;
(h) measuring the intensity of fluorescence of the supernatant; and
(i) relating the measured intensity of fluorescence to the amount of analyte in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification