Method for analyte detection using proximity probes

Inventors

Schallmeiner, Edith

Assignees

Olink Proteomics AB

Abstract

A method for detecting an analyte in a sample, comprising (a) contacting the sample with at least one set of at least first, second and third proximity probes, which probes each include an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte, the nucleic acid domain of the third proximity probe being a splint which is capable of hybridizing at least to the nucleic acid domains of the first and second proximity probes, wherein when all of the at least three proximity probes bind to the analyte, the nucleic acid domains of the first and second proximity probes are conjugatable by means of an interaction mediated by the hybridized splint of the third proximity probe; (b) conjugating the nucleic acids, of the first and second proximity probes; and (c) detecting the conjugation. Also provided is a kit for use in such a method.

Core Innovation

The document describes a proximity ligation/analyte detection assay that uses at least one set of first, second and third proximity probes, where each proximity probe comprises an analyte-binding domain and a nucleic acid domain. The nucleic acid domain of the third proximity probe is a splint capable of hybridizing at least to the nucleic acid domains of the first and second proximity probes. When all of the at least three proximity probes bind to the analyte, the nucleic acid domains of the first and second proximity probes are directly or indirectly conjugatable by means of a ligation reaction templated by the hybridized splint of the third proximity probe.

The document frames the approach as tethering templating to the bound probe complex by using the splint as a nucleic acid domain of a third proximity probe that binds the analyte. This localization of templated ligation or conjugation within the analyte-bound complex is presented as reducing background and improving sensitivity and specificity relative to free-splint two-probe assays, while still enabling conjugating the nucleic acids of the first and second proximity probes and detecting the conjugation to detect the analyte.

The document further describes assay formats and implementations including homogeneous and solid-phase formats, multiplexing, and detection approaches such as real-time PCR/qPCR and DNA array readout. It also discusses kit-level components for practicing the method and includes examples showing quantitative performance improvements for analytes including VEGF, PSA and PSA-ACT, and troponin I, as well as probe, conjugation, detection concepts and related processing elements such as blocking oligonucleotides, cassette oligonucleotide, and gap filling.

Claims Coverage

The provided independent claim set centers on a method for detecting an analyte using at least three proximity probes, where a third probe provides a splint that hybridizes to the nucleic acid domains of first and second probes to template ligation or conjugation, followed by detection of the conjugation product.

Across the independent claim structure described for this family, the core inventive concept is templated ligation or conjugation between nucleic acid domains of first and second proximity probes when co-localized by an analyte-binding third proximity probe that provides the splint, followed by detection of the resulting conjugation to detect the analyte.

Stated Advantages

Documented Applications