Claims
- 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.
- 2. The method of claim 1, wherein said predefined ratio of ligand-GFP conjugate, anti-ligand and volume is determined by obtaining a binder dilution profile comprising:
(i) providing a known volume of a solution containing a known amount of said ligand-GFP conjugate; (ii) contacting a known amount of the support and immobilized anti-ligand with said solution; (iii) incubating said solution containing ligand-GFP conjugate and immobilized anti-ligand for a predetermined time; (iv) separating the supernatant of said solution from the immobilized anti-ligand; (v) measuring the intensity of fluorescence of the supernatant; (vi) repeating steps (i)-(v) for a plurality of known amounts of ligand-GFP conjugate and immobilized anti-ligand; and (vii) selecting a test amount of immobilized anti-ligand based on said measurements, thereby determining said predefined ratio.
- 3. The method of claim 1, wherein said relating step entails obtaining a dose response profile comprising:
(i) combining a test amount of support having said anti-ligand immobilized thereon with a known volume of a solution containing a known amount of ligand-GFP conjugate; (ii) combining a known amount of non-fluorescent ligand with said test amount of support and said solution to form a mixture thereof; (iii) incubating said mixture under predetermined conditions; (iv) separating the supernatant of the mixture from the support; (v) measuring the intensity of fluorescence of the supernatant; (vi) repeating steps (i)-(v) for a plurality of amounts of the non-fluorescent ligand; and (vii) relating said measurements to the known amounts of non-fluorescent ligand.
- 4. The method of claim 1, wherein said GFP is an enhanced green fluorescent protein.
- 5. The method of claim 1, wherein in said fluorescence the excitation maxima occurs at about 380-500 nm and the emission maxima occurs at about 450-520 nm.
- 6. The method of claim 1, wherein the ligand is biotin, and the anti-ligand is avidin.
- 7. The method of claim 6, wherein said analyte is a biotinylated biomolecule.
- 8. The method of claim 7, wherein said biomolecule is selected from the group consisting of agonists, antagonists, toxins, venoms, viral epitopes, hormones, hormone receptors, polypeptides, enzymes, cofactors, enzyme substrates, drugs, lectins, sugars, oligonucleotides, oligosaccharides, proteins, and antibodies.
- 9. The method of claim 1, wherein said ligand is a hapten and said anti-ligand is an immunoglobulin having specific binding affinity for the hapten.
- 10. The method of claim 9, wherein the hapten is an oligo- or polypeptide.
- 11. The method of claim 10, wherein the oligo- or polypeptide is covalently linked to the GFP label as a fusion protein.
- 12. The method of claim 9, wherein said analyte is a non-fluorescent antigen having a specific binding affinity for the anti-ligand.
- 13. The method of claim 1, wherein said ligand is an antibody and said anti-ligand is a hapten for said antibody.
- 14. The method of claim 13, wherein said analyte is a non-fluorescent antibody having a specific binding affinity for the anti-ligand.
STATEMENT OF GOVERNMENT SUPPORT
[0001] Development of the present invention was supported in part by the National Institutes of Health (Grant GM47915) and the Department of Energy (Grant DE-FG05-95ER62010). The Government may have certain rights in the invention.