SAMPLE CLARIFICATION AND REDUCTION OF BACKGROUND FLUORESCENCE FOR FLUORESCENT DETECTION OF ANALYTES

• US 20190324029A1
• Filed: 04/19/2019
• Published: 10/24/2019
• Est. Priority Date: 04/19/2018
• Status: Active Grant

• Current Assignee: Ellie Company
• Original Assignee: Ellie Company
• Inventors: Kostovic, Miladin, Stojanovic, Milovan
• Granted Patent: US 11,435,342 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/743 Steroid hormones

Abstract

Methods and reagents for processing samples for fluorescence analysis. Processing methods include treating samples containing riboflavin to reduce riboflavin-dependent autofluorescence by adding riboflavin binding protein to the sample, irradiating the sample, or a combination thereof. Processing methods also include clarifying samples by coagulating, precipitating, and/or otherwise removing proteins and other components that interfere with fluorescence analysis without removing the analyte. Fluorescence analysis methods include fluorescence polarization analysis (FPA) and others. Reagents suitable for performing the disclosed methods are provided.

Claims

1. A method of treating and testing a sample comprising an analyte, the method comprising:

treating the sample to reduce riboflavin-dependent autofluorescence in the sample, wherein the treating comprises adding riboflavin binding protein to the sample, irradiating the sample, or a combination thereof;

adding a tracer to the sample; and

detecting fluorescence from the tracer in the sample, wherein the detected fluorescence indicates an amount of analyte in the sample.

2. The method of claim 1, wherein the treating comprises adding riboflavin binding protein to the sample.

3. The method of claim 2, wherein the detecting is performed in the presence of the riboflavin binding protein added to the sample.

4. The method of claim 1, wherein the treating comprises irradiating the sample.

5. The method of claim 1, further comprising clarifying the sample while maintaining the analyte in the sample.

6. The method of claim 5, wherein the clarifying comprises coagulating casein in the sample and removing the coagulated casein from the sample.

7. The method of claim 6, wherein the coagulating comprises adding a rennet to the sample in an amount sufficient to coagulate the casein.

8. The method of claim 5, wherein the clarifying comprises removing lipid from the sample.

9. The method of claim 5, wherein the clarifying further comprises adding a non-aqueous polar solvent to the sample.

10. The method of claim 9, wherein the non-aqueous polar solvent comprises an alcohol.

11. The method of claim 9, wherein the non-aqueous polar solvent is added to the sample in an amount sufficient to maintain the analyte in the sample.

12. The method of claim 9, wherein the non-aqueous polar solvent is added to the sample at a final concentration of from about 10% v/v to about 50% v/v.

13. The method of claim 5, wherein the clarifying comprises centrifuging the sample.

14. The method of claim 5, wherein the clarifying comprises filtering the sample.

15. The method of claim 14, wherein the filtering is conducted with a filter having pore size from about 0.1 μ

m to about 10 μ

m.

16. The method of claim 1, wherein the sample comprises a milk product.

17. The method of claim 1, wherein the sample comprises milk serum.

18. The method of claim 1, wherein the analyte is a hydrophobic analyte.

19. The method of claim 1, wherein the analyte is progesterone.

20. The method of claim 1, wherein the analyte comprises an antibody or an antigen.

21. The method of claim 1, wherein the tracer comprises a binding moiety and a fluorescent moiety, wherein the binding moiety comprises a standard of the analyte or a ligand of the analyte.

22. The method of claim 1, further comprising, prior to the detecting, adding to the sample a ligand to which the analyte and the tracer compete for binding.

23. The method of claim 1, wherein the detecting comprises detecting plane-polarized light.