Methods for the Analysis of Proximity Binding Assay Data

• US 20110208441A1
• Filed: 08/05/2010
• Published: 08/25/2011
• Est. Priority Date: 08/05/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Shiaw-Min, Leong, Harrison M., Ruff, David W.
• Granted Patent: US 10,208,335 B2
• Time in Patent Office: Days
• US Class Current: 702/32
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR

Abstract

Various embodiments of methods for analyzing proximity binding assay (PBA) data are disclosed. Proximity binding assays as a class of analyses offer the advantages of the sensitivity and specificity of biorecognition binding, along with the exponential signal amplification offered by a variety of oligonucleotide amplification reactions, such as the polymerase chain reaction (PCR). However, as various proximity binding assays have reaction kinetics governed by an additional step of the binding of a biorecognition probe (BRP) with a target molecule, there is a need for methods for the analysis of PBA data that are particularly suited to the unique characteristics of such data.

Claims

1. A system for analyzing proximity binding assay data, comprising:

a thermal cycler instrument; and

a processor in communication with the thermal cycler instrument that

receives proximity binding assay data for a plurality of samples, wherein the proximity binding assay data comprises at least one set of test sample data and at least one set of reference sample data;

determines cycle threshold (Ct) values for the at least one set of test sample data and at least one set of reference sample data;

calculates background corrected Ct values for each value in the test sample data set and the reference sample data set with a corresponding value in a background data set;

determines the linear range for the background corrected Ct values as a function of sample quantity for each set of test sample data and reference sample data; and

selects a background corrected Ct value as a threshold value;

where after the steps of determining the linear range for each set of test and reference data, and selecting a corrected Ct threshold, a quantity for a target molecule in the at least one test sample is determined.

2. The system of claim 1, further comprising a processor in communication with the thermal cycler instrument that determines a confidence value for the quantity determined for the target molecule in the at least one test sample.

3. The system of claim 1, wherein background data set is a negative control in which the target molecule of the at least one test sample is absent.

4. The system of claim 1, where the quantity determined for the target molecule in the at least one test sample is relative.

5. The system of claim 1, where the quantity determined for the at least one test sample is quantitative.

6. The system of claim 1, where the target molecule is a protein.

7. The system of claim 1, where the at least one test sample is a cell sample.

8. A method for analyzing proximity binding assay data, the method comprising:

providing proximity binding assay data for a plurality of samples, wherein the proximity binding assay data comprises at least one set of test sample data and at least one set of reference sample data;

determining cycle threshold (Ct) values for the at least one set of test sample data and at least one set of reference sample data;

calculating background corrected Ct values for each value in the test sample data set and the reference sample data set with a corresponding value in a background data set;

determining the linear range for the background corrected Ct values as a function of sample quantity for each set of test sample data and reference sample data; and

selecting a background corrected Ct value as a threshold value;

where after the steps of determining the linear range for each set of test and reference data, and selecting a corrected Ct threshold, a quantity for a target molecule in the at least one test sample is determined.

9. The method of claim 8, further comprising determining a confidence value for the quantity determined for the target molecule in the at least one test sample.

10. The method of claim 8, where the background data set is a negative control in which the target molecule of the at least one test sample is absent.

11. The method of claim 8, where the quantity determined for the target molecule in the at least one test sample is relative.

12. The method of claim 8, where the quantity determined for the at least one test sample is quantitative.

13. The method of claim 8, where the target molecule is a protein.

14. The method of claim 8, where the at least one test sample is a cell sample.

15. A computer-readable medium and computer-readable code embodied on said computer-readable medium for analyzing proximity assay binding data, the computer-readable code comprising:

obtaining proximity binding assay data for a plurality of samples, wherein the proximity binding assay data comprises at least one set of test sample data and at least one set of reference sample data;

determining cycle threshold (Ct) values for the at least one set of test sample data and at least one set of reference sample data;

calculating background corrected Ct values for each value in the test sample data set and the reference sample data set with a corresponding value in a background data set;

determining the linear range for the background corrected Ct values as a function of sample quantity for each set of test sample data and reference sample data; and

selecting a background corrected Ct value as a threshold value;

where after the steps of determining the linear range for each set of test and reference data, and selecting a corrected Ct threshold, a quantity for a target molecule in the at least one test sample is determined.

16. The computer-readable medium of claim 15, further comprising determining a confidence value for the quantity determined for the target molecule in the at least one test sample.

17. The computer-readable medium of claim 15, where the background data set is a negative control in which the target molecule of the at least one test sample is absent.

18. The computer-readable medium of claim 15, where the quantity determined for the target molecule in the at least one test sample is relative.

19. The computer-readable medium of claim 15, where the quantity determined for the at least one test sample is quantitative.

20. The computer-readable medium of claim 15, where the target molecule is a protein.

21. The computer-readable medium of claim 15, where the at least one test sample is a cell sample.