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US 11,447,815 B2Granted
Methods for the analysis of proximity binding assay data
AssigneeLife Technologies Corporation
InventorsShiaw-Min Chen, David W. Ruff, Harrison Leong
Life of this patent
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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.
Claims14
A method of using a proximity binding assay to determine the quantity of a target molecule in test samples, the method comprising:
- adding a first biorecognition probe modified with a first oligonucleotide sequence and a second biorecognition probe modified with a second oligonucleotide sequence to a first sample region comprising the test samples, a second sample region comprising reference samples, and a third sample region comprising negative control samples and lacking the target molecule, the first and second biorecognition probes being designed to readily attach to the target molecule, when present, and the first oligonucleotide sequence and the second oligonucleotide sequence being designed to readily bind when in proximity to each other, resulting in a target for amplification;
- obtaining, from the first sample region, the second sample region, and the third sample region, any targets for amplification resulting from adding;
- respectively combining any targets for amplification obtained from the first sample region, the second sample region, and the third sample region with labeling probes;
- conducting thermal cycling on respective combinations comprising the targets for amplification and the labeling probes;
- obtaining proximity binding assay data from detecting the labeling probes during or after thermal cycling, the proximity binding assay data comprising test sample data, reference sample data and negative control sample data;
- and
- processing the proximity binding assay data to determine a quantity of the target molecule in each of the test samples, wherein processing comprises using the negative control sample data to correct for background binding of the first oligonucleotide sequence and the second oligonucleotide sequence that occurs even without presence of the target molecule in a sample.
4 more elements
A kit for determining the quantity of a target molecule in a test samples, the kit comprising:
- a plurality of first biorecognition probes and a plurality of second biorecognition probes, a first biorecognition probe of the plurality of first biorecognition probes being modified with a first oligonucleotide sequence and a second biorecognition probe of the plurality of second biorecognition probes being modified with a second oligonucleotide sequence, the first biorecognition probe and the second biorecognition probe being designed to readily attach to the target molecule, when present, and the first oligonucleotide sequence and the second oligonucleotide sequence being designed to readily bind when in proximity to each other, resulting in a target for amplification;
- and
- a computer program product embedded in a non-transitory computer readable medium comprising executable instruction code that, when executed by one or more processors causes the one or more processors to perform processing comprising:
- processing proximity binding assay data to determine a quantity of the target molecule in the test samples;
- wherein:
- the proximity binding assay data comprises test sample data, reference sample data, and negative control sample data;
- the test sample data has been obtained by detecting labeling probes during or after thermocycling of a combination comprising the labeling probes and any obtained targets for amplification, the obtained targets for amplification having been obtained from adding the first and second biorecognition probes to the test samples;
- the reference sample data has been obtained by detecting labeling probes during or after thermocycling of a combination comprising the labeling probes and any obtained targets for amplification, the obtained targets for amplification having been obtained from adding the first and second bio recognition probes to reference samples;
- the negative control sample data has been obtained by detecting labeling probes during or after thermocycling of a combination comprising the labeling probes and any obtained targets for amplification, the obtained targets for amplification having been obtained from adding the first and second bio recognition probes to negative control samples in which the target molecule is not present;
- and
- processing proximity binding assay data comprises using the negative control sample data to correct for background binding of the first oligonucleotide sequence and the second oligonucleotide sequence that occurs even without presence of the target molecule in a sample.
8 more elements
Dependencies are inferred from the claim text.