Longitudinal assay

• US 8,975,087 B2
• Filed: 11/30/2010
• Issued: 03/10/2015
• Est. Priority Date: 11/24/2010
• Status: Active Grant

• Current Assignee: Inanovate, Inc.
• Original Assignee: Inanovate, Inc.
• Inventors: Nelson, James Curtis, Ure, David
• Primary Examiner(s): Counts, Gary W
• Application Number:
  US12/956,117
• Publication Number:
  US 20120129271A1
• Time in Patent Office: 1,561 Days
• Field of Search: None
• US Class Current: 436/518
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: F04B 43/0009 Special features
• CPC Class Codes: F04B 43/02 having plate-like flexible members, e.g. diaphragms F04B43/14 takes precedence
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/557 using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction

Abstract

Embodiments of the invention include methods for detecting and determining the concentrations of macro and small molecules, including bio-molecules, in a liquid or gaseous sample.

Claims

1. A method of detecting and determining a concentration of a labeled analyte in a fluid sample, the method comprising:

passing the fluid sample containing the labeled analyte across at least one assay surface containing a capture agent for the labeled analyte;

detecting the labeled analyte and determining a first detection intensity at a first temporal point;

repeating the passing and detecting steps at least once using the same fluid sample and determining at least one additional detection intensity at at least one additional temporal point;

creating a binding curve for the labeled analyte including the first and at least one additional detection intensities; and

determining a concentration of the analyte in the fluid sample based on a comparison of the first detection intensity and the at least one additional detection intensity in the binding curve.

2. The method of claim 1, wherein the analyte and the capture agent are each independently selected from a group consisting of:

a protein;

a peptide;

an antibody;

an auto-antibody;

an antigen;

a native antigen;

a protein complex comprising a complex of a protein and at least one of the following;

an antibody, an antigen, a native antigen, or an auto-antibody;

a lipid;

DNA; and

RNA; and

wherein the capture agent may further comprise a cell or tissue lysate or fractions thereof.

3. The method of claim 1, wherein the labeled analyte is labeled with at least one of the following:

a fluorescent marker, a luminescent marker, a colormetric marker, or a radioactive marker.

4. The method of claim 1, wherein the fluid sample is a liquid.

5. The method of claim 1, wherein the detecting occurs during the passing of the fluid sample.

6. The method of claim 1, wherein the detecting occurs after the passing of the fluid sample.

7. The method of claim 1, wherein repeating the passing and detecting steps at least once includes repeating the passing and detecting steps between about 5 times and about 20 times.

8. The method of claim 1, further comprising:

distinguishing specific binding from non-specific binding in the detected labeled analyte using the binding curve and/or a constant associated with the binding curve.

9. The method of claim 8, wherein the distinguishing specific binding from non-specific binding comprises:

determining a first detection intensity and a first temporal point at which a rise to an equilibrium may be inferred; and

attributing to non-specific binding a portion of at least one detected intensity subsequent to the first temporal point that is equal to the first detection intensity.

10. The method of claim 9, wherein distinguishing specific binding from non-specific binding further includes:

subtracting from a second detection intensity the value of the first detection intensity; and

attributing to specific binding a remaining portion of the second detection intensity,

wherein the second detection intensity is greater than the first detection intensity and the second detection intensity occurs at a second temporal point that is subsequent to the first temporal point.

11. The method of claim 8, wherein distinguishing specific binding from non-specific binding includes:

determining a first detection intensity and a first temporal point at which a rise to an equilibrium has been reached; and

attributing the first detection intensity to non-specific binding.

12. The method of claim 11, wherein distinguishing specific binding from non-specific binding further includes:

attributing detection intensities occurring subsequent to the first temporal point to non-specific binding; and

concluding that the detection intensities of the binding curve do not include a specific binding component.

14. The method of claim 1, further comprising:

passing a non-labeled solution across the at least one assay surface.

13. A method of detecting and determining a concentration of an analyte in a fluid sample, the method comprising:

passing the fluid sample containing the analyte across at least one assay surface containing a capture agent for the analyte;

passing a solution containing a labeled molecule across the at least one assay surface to label at least a portion of the total analyte in the fluid sample, the labeled molecule being capable of binding to the analyte;

passing a non-labeled solution across the at least one assay surface;

detecting the labeled molecule and determining a first detection intensity at a first temporal point;

repeating the passing steps and the detecting at least once using the same fluid sample and determining at least one additional detection intensity at at least one additional temporal point;

creating a binding curve for the analyte including the first and at least one additional detection intensities; and

determining a total concentration of the labeled and unlabeled analyte in the fluid sample based on a comparison of the first detection intensity and the at least one additional detection intensity in the binding curve of the labeled molecule.

15. A method of detecting and determining a concentration of an analyte in a fluid sample, the method comprising:

passing the fluid sample containing the analyte across at least one assay surface containing a capture agent for the analyte;

passing a solution containing a labeled molecule across the at least one assay surface, the labeled molecule being capable of binding to the analyte;

detecting the labeled molecule and determining a first detection intensity at a first temporal point;

repeating each of the passing steps and the detecting step at least once and determining at least one additional detection intensity at at least one additional temporal point;

creating a binding curve for the analyte including the first and at least one additional detection intensities; and

determining a concentration of the analyte in the fluid sample based on a comparison of the first detection intensity and the at least one additional detection intensity in the binding curve.

16. The method of claim 15, further comprising:

passing a non-labeled solution across the at least one assay surface after passing the fluid sample across the at least one assay surface.

17. The method of claim 15, further comprising:

passing a non-labeled solution across the at least one assay surface after passing the solution containing the labeled molecule across the at least one assay surface.