Assay with increased dynamic range

• US 9,157,910 B2
• Filed: 03/15/2013
• Issued: 10/13/2015
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Dowell, Barry L., Gayda, Susan, Ruan, Qiaoqiao, Skinner, Joseph P., Tetin, Sergey Y.
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US13/833,655
• Publication Number:
  US 20140273035A1
• Time in Patent Office: 942 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Abstract

Provided herein are assays and kits useful for avoiding “prozone phenomenon” or “hook effect” and which expand the range of accurately measurable analyte concentrations.

Claims

1. A kit comprising:

i) a first analyte-binding molecule comprising a first label;

ii) a second analyte-binding molecule comprising a second label, wherein the binding affinity for the analyte of the first analyte-binding molecule is greater than that of the second analyte-binding molecule;

iii) a third analyte-binding molecule attached to a solid support, wherein the third analyte-binding molecule can bind to analyte concurrently with either the first analyte-binding molecule or the second analyte-binding molecule;

wherein the solid support comprises two or more spatially separated electrodes, wherein the solid support is contained in a handheld point-of-care device.

2. The kit of claim 1, wherein one or more of the first analyte-binding molecule, the second analyte-binding molecule, and/or the third analyte-binding molecule is an antibody or fragment thereof.

3. The kit of claim 1, wherein the first analyte-binding molecule and the second analyte-binding molecule are directly attached to the label.

4. The kit of claim 3, wherein the solid support is selected from the group consisting of a particle, a microparticle, a bead, an electrode, and a multiwell plate.

5. The kit of one of claim 4, wherein the solid support comprises a microparticle.

6. The kit of claim 1, wherein one or both of the first label and the second label are selected from the group consisting of an enzyme, a chromophore, and a fluorophore.

7. The kit of claim 1, wherein the first label and the second label are different.

8. The kit of claim 1, wherein the difference in binding affinity of the first analyte-binding molecule and the second analyte-binding molecule for the analyte ranges from about 5-fold to about 100-fold.

9. The kit of claim 1, wherein the difference in binding affinity of the first analyte-binding molecule and the second analyte-binding molecule for the analyte is at least about 100-fold.

10. The kit of claim 1, wherein said kit can be employed for a one-step sandwich assay.

11. A method of expanding the dynamic range of an assay, comprising:

a) contacting a test sample suspected of comprising an analyte with a first analyte-binding molecule comprising a first label, a second analyte-binding molecule comprising a second label and a third analyte-binding molecule attached to a solid support under conditions that allow binding of;

(i) the first analyte-binding molecule and the third analyte-binding molecule and

(ii) the second analyte-binding molecule and the third analyte-binding molecule

to the analyte, wherein the binding affinity for the analyte of the first analyte-binding molecule is greater than that of the second analyte-binding molecule, wherein the first analyte-binding molecule and the second analyte-binding molecule do not concurrently bind to the analyte;

b) measuring the signal intensities of the first label of the first analyte-binding molecule bound to the analyte and of the second label of the second analyte-binding molecule bound to the analyte; and

c) determining the concentration of analyte by comparing the signal intensities of the first label and the second label, wherein step b) of measuring the signal intensities of the first label of the first analyte-binding molecule bound to the analyte and the signal intensity of the second label of the second analyte-binding molecule bound to the analyte is done in a calibration assay over a predetermined range of analyte concentrations, and the method further comprises the step of;

d) establishing a flag value by determining a ratio of the signal intensity of the first label of the first analyte-binding molecule bound to the analyte and the signal intensity of the second label of the second analyte-binding molecule bound to the analyte in the calibration assay or the inverse of this ratio at or near the concentration of analyte that provides maximum signal intensity of the first label of the first analyte-binding molecule bound to the analyte.

12. The method of claim 11, wherein one or more of the first analyte-binding molecule, the second analyte-binding molecule, and/or the third analyte-binding molecule is an antibody or fragment thereof.

13. The method of claim 11, wherein the first analyte-binding molecule and the second analyte-binding molecule are directly attached to the label.

14. The method of claim 11, wherein the solid support is selected from the group consisting of a particle, a microparticle, a bead, an electrode and a multiwell plate.

15. The method of claim 11, wherein one or both of the first label and the second label are selected from the group consisting of an enzyme, a chromophore, and a fluorophore.

16. The method of claim 11, wherein the first analyte-binding molecule and the second analyte-binding molecule are contacted with the test sample in the same reaction mixture.

17. The method of claim 11, wherein the first analyte-binding molecule and the second analyte-binding molecule are contacted with the test sample in the different reaction mixtures.

18. The method of claim 17, wherein the first label and the second label are the same.

19. The method of claim 11, wherein the first label and the second label are different.

20. The method of claim 11, wherein the difference in binding affinity of the first analyte-binding molecule and the second analyte-binding molecule for the analyte ranges from about 5-fold to about 100-fold.

21. The method of claim 11, wherein the difference in binding affinity of the first analyte-binding molecule and the second analyte-binding molecule for the analyte is at least about 100-fold.

22. The method of claim 11, wherein the dynamic range of the assay comprises three or more orders of magnitude.

23. The method of claim 11, wherein the first analyte-binding molecule and the second analyte-binding molecule are present in predetermined molar amounts that differ by less than about 100-fold.

24. The method of claim 11, wherein the first analyte-binding molecule and the second analyte-binding molecule are not oligomerized or cross-linked.

25. The method of claim 11, wherein when the ratio of the signal intensity of the second label of the second analyte-binding molecule bound to the analyte to the signal intensity of the first label of the first analyte-binding molecule bound to the analyte in the test sample:

exceeds or equals the flag value, then the sinking section of the calibration curve from the signal intensity of the first label of the first analyte-binding molecule bound to the analyte is used to determine analyte concentration;

or

is less than the flag value, then the rising section of the calibration curve from the signal intensity of the first label of the first analyte-binding molecule bound to the analyte is used to determine analyte concentration.

26. The method of claim 11, wherein when the ratio of the signal intensity of the first label of the first analyte-binding molecule bound to the analyte to the signal intensity of the second label of the second analyte-binding molecule bound to the analyte in the test sample:

is less than or equals the flag value, then the sinking section of the calibration curve from the signal intensity of the first label of the first analyte-binding molecule bound to the analyte is used to determine analyte concentration;

or

exceeds the flag value, then the rising section of the calibration curve from the signal intensity of the first label of the first analyte-binding molecule bound to the analyte is used to determine analyte concentration.

27. The method of claim 11, wherein the method is performed using an automated or semi-automated system.

28. The method of claim 11, wherein the assay is a one step assay.