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Publication Number
US-9677131-B2
Publication Date
2017-06-13
Expiration Date
Abstract
The present invention relates to the use of a conjugate of a non-analyte-specific binding protein coupled to a nucleic acid as a blocking reagent in a probe-based detection assay, which uses a probe comprising a proteinaceous analyte-binding partner coupled to a nucleic acid domain to detect an analyte in a sample.
Core Innovation
The invention relates to a kit for use in a method for detecting an analyte in a sample using at least one set of proximity probes and generating a nucleic acid signal. Each proximity probe in a set comprises a proteinaceous analyte-specific binding domain and a nucleic acid domain. The method is described in the context of proximity-probe based detection assays, including proximity ligation assay (PLA) formats and related nucleic-acid interaction assays.
A core problem addressed is non-specific background signal originating from random probe proximity. The invention provides a blocking reagent designed to mimic proximity probes so that non-analyte-specific binding sites are occupied while reducing non-specific background, without significantly reducing analyte. The blocking reagent comprises a non-analyte-specific binding protein conjugated to a nucleic acid domain, and is used in excess.
The blocking reagent is characterized by a non-analyte-specific binding protein with negligible specific affinity to the analyte and optionally exemplified by serum proteins such as bulk IgG, IgG/γ-globulin, or albumin (BSA/HSA), as well as streptavidin/streptavidin-like proteins. The blocking reagent nucleic acid domain is designed with sequence constraints relative to probe/splint/other oligonucleotide nucleic acids, including less than 80% sequence identity, and can optionally include randomly generated sequences. The document further describes that the kit can be used across different proximity-probe assay formats and includes an experimental demonstration that conjugated blocking reagent reduces mismatched PLA background versus individual unconjugated components.
Claims Coverage
The independent claim provides the overall kit structure using a blocking reagent and analyte-specific proximity probes. Dependent claim coverage refines the non-analyte-specific binding protein, specifies nucleic acid design constraints for the blocking reagent, and narrows aspects of conjugation and use in proximity-probe workflows that result in ligation or extension detection.
Blocking reagent as protein–nucleic acid conjugate
A kit comprising a blocking reagent comprising a non-analyte-specific binding protein conjugated to a nucleic acid domain.
Analyte-specific proximity probe sets with proteinaceous binding and nucleic acids
At least one set of probes, wherein each probe in a set comprises a proteinaceous analyte-specific binding domain and a nucleic acid domain.
Serum globulin composition constraints
The non-analyte-specific binding protein is derived from blood serum globulin comprising at least 70% γ-globulins.
Streptavidin-like non-analyte-specific binding protein
The non-analyte-specific binding protein is a streptavidin-like protein with similar structural and/or functional properties to streptavidin, or a modification/derivative/variant thereof.
Nucleic acid sequence identity and strand constraints for the blocking reagent
The blocking reagent nucleic acid domain has less than 80% sequence identity to probe/splint/other oligonucleotide nucleic acids, and may include randomly generated sequences, at least 8 nucleotides, and/or be single-stranded DNA.
Covalent linkage between blocking protein and nucleic acid
The non-analyte-specific binding protein is conjugated to the nucleic acid by covalent linkage.
Proximity probe interaction enabling ligation or extension detection
The method comprises contacting the sample with the blocking reagent, contacting with first/second proximity probes that simultaneously bind the analyte, allowing nucleic acid interaction via ligation or extension, and detecting the ligation/extension.
Across the independent claim and refinements, the inventive concept is a kit that uses a conjugated blocking reagent made from a non-analyte-specific binding protein and a nucleic acid domain together with analyte-specific proximity probes having proteinaceous binding domains and nucleic acid domains. Dependent claims narrow the specific non-analyte-specific binding protein types, impose sequence/strand identity constraints on the blocking nucleic acid domain, restrict conjugation to covalent linkage, and define use patterns that lead to ligation or extension detection.
Stated Advantages
Reduces non-specific background signal resulting from random probe proximity.
Occupies non-specific binding sites while reducing background.
Reduces mismatched PLA background versus individual unconjugated components.
Blocking works across species and across different conjugation chemistries.
Documented Applications
Proximity-probe based detection assays including proximity ligation assay (PLA).
Ligation and hybridization/extension proximity-probe assay formats.
Padlock/RCA variants of proximity-probe based detection.
Immuno-PCR and immuno-RCA contexts.
Use in detecting an analyte in a sample with proximity probes and nucleic acid signal generation.
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