Patent ID: 7932060

Claim:
A method of detecting an analyte in a sample, comprising: (i) combining: (a) an analyte; (b) a first proximity member, comprising a first analyte-specific binding entity and a single stranded first oligonucleotide comprising a first portion wherein the first analyte-specific binding entity is capable of forming a complex with the analyte and is conjugated to the first oligonucleotide; (c) a second proximity member, comprising a second analyte-specific binding entity and a single stranded second oligonucleotide comprising a portion that is capable of hybridizing to the first portion of the first oligonucleotide wherein the second analyte-specific binding entity is capable of forming a complex with the analyte and is conjugated to the second oligonucleotide; and (d) a hybridization blocker oligonucleotide at a concentration in excess of the concentrations of the first and second proximity members, wherein the hybridization blocker oligonucleotide comprises a portion that is capable of forming a hybrid with the first portion of the first oligonucleotide to reduce hybridization between the first and second oligonucleotides and wherein the first analyte specific binding entity and/or the second analyte specific binding entity is a protein or a protein complex; (ii) forming a complex comprising the analyte, the first proximity member, the second proximity member, and the hybridization blocker oligonucleotide that is hybridized with the first portion of the first oligonucleotide; (iii) forming a hybrid by displacing the hybridization blocker oligonucleotide from the first portion of the first oligonucleotide of the complex wherein the hybrid comprises the first portion of the first oligonucleotide and the portion of the second oligonucleotide, such that the 3′ terminus of the first oligonucleotide and/ or second oligonucleotide of the hybrid can be extended via a polymerase; (iv) extending via a polymerase the 3′ terminus of the first oligonucleotide and/ or second oligonucleotide and producing an amplicon; (v) amplifying the amplicon and producing an amplification product; and (vi) detecting the amplification product, wherein the detection of the amplification product indicates the presence of the analyte in the sample.