Kit for proximity probing with multivalent proximity probes
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-8013134-B2
Publication Date
2011-09-06
Expiration Date
Abstract
The present invention relates to sensitive, rapid and convenient assays for detection and or quantification of one or more analyte(s) in solution using multivalent proximity probes. The proximity probes each comprise several binding moieties, such as antibodies, and associated nucleic acid(s). When the binding moieties have bound to their analyte(s), the nucleic acids on opposite proximity probes interact with each other and a signal is generated based on this interaction. The multivalent proximity probes are especially valuable for highly sensitive and specific protein detection.
Core Innovation
The invention relates to proximity ligation/proximity probing in solution and describes detection or quantification of an analyte using nucleic-acid ligation when proximity is achieved, with detection based on the nucleic-acid product. Assay sensitivity depends on binding affinity and background ligation from probe concentrations, and the approach enables interaction of nucleic acids when in close proximity and contact.
A core concept is the use of multivalent proximity probes to increase effective affinity. Each multivalent proximity probe includes multiple binding moieties linked via flexible linkers to reactive nucleic acids, and multiple copies of an analyte can be bound by more than one binding moiety on one or more probes, including sandwich-like sandwiched targets, enabling ligation and subsequent nucleic-acid amplification for detection or quantification.
The disclosure also provides kit compositions for multivalent proximity probes with reactive nucleic acids and optional components such as a splint ligation template, ligase, and PCR primers, with optional coupling such as biotin/streptavidin. It further describes universal/probe strategies and applications including multiplex detection, competitive assays, and antagonist screening, and includes construction concepts such as polymer backbones and nucleic-acid backbone/aptamer concatemer options.
Claims Coverage
The provided independent claims are directed to kits that implement proximity-dependent ligation using a pair of different streptavidin-oligonucleotide conjugates with complementary ends, together with a splint ligation template oligonucleotide. The independent claim set further covers optional amplification support via PCR primers, yielding detectable new nucleic acid product(s).
Proximity-based splint ligation using streptavidin-oligonucleotide conjugates
A kit comprising a pair of different streptavidin-oligonucleotide conjugates, each comprising an oligonucleotide and streptavidin not associated with a biotinylated molecule, where one conjugate has an oligonucleotide with a free 3′ end and the other has an oligonucleotide with a free 5′ end, and a splint ligation template oligonucleotide having a sequence that can hybridize to both oligonucleotides when the oligonucleotides are in proximity to one another and in contact with the splint ligation template oligonucleotide.
PCR primer-supported amplification of proximity ligation products
A kit comprising a pair of different streptavidin-oligonucleotide conjugates where the oligonucleotide in one conjugate is different from the oligonucleotide in the other, a splint ligation template oligonucleotide having a sequence that can hybridize to both oligonucleotides when the oligonucleotides are in proximity and in contact with the splint ligation template oligonucleotide, and a pair of PCR primers, one hybridizing to the oligonucleotide of one conjugate and the other hybridizing to the oligonucleotide of the other conjugate.
Across the independent claims, the core coverage is a proximity ligation kit architecture using two different streptavidin-oligonucleotide conjugates and a splint ligation template that supports ligation when the conjugate oligonucleotides are in proximity and contact. One independent claim specifies free 3′/5′ ends in the conjugate oligonucleotides, and the other specifies different oligonucleotide sequences on the conjugates together with PCR primers that hybridize to those sequences for amplification.
Stated Advantages
Improved detection of human insulin with multivalent streptavidin/biotin networks.
Extension of detection to VEGF using polyclonal anti-VEGF antibodies.
Documented Applications
Multiplex detection.
Competitive detection.
Screening ligand/receptor antagonists.
Detection of human insulin.
Detection of VEGF (vascular endothelial growth factor).
Proximity probing/proximity ligation assay in solution.
Detection or quantification based on nucleic-acid product via amplification.
Interested in licensing this patent?