Assay with internal calibration

• US 9,005,901 B2
• Filed: 03/15/2013
• Issued: 04/14/2015
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Ruan, Qiaoqiao, Skinner, Joseph P., Tetin, Sergey Y., Gayda, Susan
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US13/833,365
• Publication Number:
  US 20140273272A1
• Time in Patent Office: 760 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 35/00693 Calibration

Abstract

Provided herein are kits and methods for assays with internal calibration.

Claims

1. A method of internally calibrating an assay, comprising:

a) contacting a capture analyte-binding molecule with a predetermined concentration of tracer analyte labeled with a first label and a predetermined concentration of detection analyte-binding molecule labeled with a second label, wherein the capture analyte-binding molecule and the detection analyte-binding molecule concurrently bind to the tracer analyte;

b) measuring the signal intensities obtained of the first label (I₁⁰) and of the second label (I₂⁰);

c) determining a correction factor (F) as the ratio of the intensity of the second label (I₂⁰) to the intensity of the first label (I₁⁰); and

d) internally calibrating the assay by dividing the predetermined concentration of tracer analyte by the correction factor (F).

3. The method of claim 1, wherein the predetermined concentration of tracer analyte is below saturation of the binding capacity of the capture analyte-binding molecule.

5. The method of claim 1, wherein a two-step assay format is used.

6. The method of claim 5, wherein when a two-step assay format is used, the predetermined concentration of detection analyte-binding molecule equals or exceeds the concentration of analyte and tracer analyte captured in the first step.

7. The method of claim 1, wherein a one-step assay format is used.

8. The method of claim 7, wherein when a one-step assay format is used, the predetermined concentration of detection analyte-binding molecule exceeds the concentration of analyte and tracer analyte.

9. The method of claim 1, wherein the method is performed using an automated or semi-automated system.

10. The method of claim 1, wherein the capture analyte-binding molecule is attached to a solid support.

11. The method of claim 10, wherein the solid support is selected from the group consisting of a particle, a microparticle, a bead, an electrode and a multiwell plate.

12. The method of claim 1, wherein one or both of the capture analyte-binding molecule and/or the detection analyte-binding molecule is an antibody or fragment thereof.

13. The method of claim 1, wherein the first label is a first chromophore and the second label is a second chromophore.

14. The method of claim 13, wherein the first label is a first fluorophore and the second label is a second fluorophore.

2. A method of determining the concentration of analyte in one or more test samples, comprising:

a) contacting a capture analyte-binding molecule with a predetermined concentration of tracer analyte labeled with a first label and a predetermined concentration of detection analyte-binding molecule labeled with a second label in either the absence or presence of one or more test samples that may comprise test analyte, wherein the capture analyte-binding molecule and the detection analyte-binding molecule concurrently bind to the tracer analyte and, if present, to test analyte in said one or more test samples;

b) measuring the signal intensities obtained in the absence of said one or more test samples of the first label (I₁⁰) and the second label (I₂⁰);

c) determining a correction factor (F) as the ratio of the intensity of the second label (I₂⁰) to the intensity of the first label (I₁⁰);

d) measuring the signal intensities obtained in the presence of said one or more test samples of the first label (I₁) and the second label (I₂); and

e) determining the concentration of test analyte in said one or more test samples by multiplying the ratio of the intensity of the second label (I₂) to the intensity of the first label (I₁) by the predetermined concentration of tracer analyte divided by the correction factor (F) and subtracting the predetermined concentration of tracer analyte;

4. The method of claim 2, wherein the predetermined concentration of tracer analyte ranges from less than the expected test analyte concentration to about 5-fold higher than the expected targeted test analyte concentration.

15. The method of claim 2, wherein steps b) and d) can be done at the same time, or in any order.

16. The method of claim 2, wherein steps d) and e) are repeated one or more times.

17. The method of claim 2, wherein steps a) through c) are performed only once.