Assay methods

A kit for the measurement of an analyte of interest in a sample, the kit comprising:

• a. a surface comprising at least one binding domain to which are bound a binding reagent for the analyte and an anchoring reagent comprising an anchoring sequence complementary to a first region of an amplicon sequence;
• and
• b. in one or more containers, compartments, or vessels:
• i. two detection reagents for the analyte, wherein the two detection reagents comprise a first proximity probe sequence and a second proximity probe sequence, respectively;
• ii. one or more connector oligonucleotides that comprise a first connector probe sequence complementary to a first region of the first proximity probe sequence and a first region of the second proximity probe sequence, and a second connector probe sequence complementary to a second non-overlapping region of the first proximity probe sequence and a second non-overlapping region of the second proximity probe sequence, wherein hybridization of the one or more connector oligonucleotides to the first and second proximity probe sequences via the complementary sequences allows ligation of the one or more connector oligonucleotides to form a circular oligonucleotide that serves as a template for producing an amplicon comprising a first region complementary to the anchoring sequence;
• and
• iii. one or more detection probes comprising a sequence complementary to a second region of the amplicon sequence, wherein each of the one or more detection probes comprise a detectable label.