Assay methods

A method of detecting a plurality of analytes of interest in a sample comprising:

• a. Contacting the sample with a surface comprising a plurality of discrete binding domains, each binding domain comprising (i) a binding reagents for an analyte, and (ii) an anchoring reagent comprising an anchoring sequence complementary to an amplicon sequence, wherein the contacting step forms a plurality of surface-bound complexes;
• b. Contacting each of the surface-bound complexes with two detection reagents for the analyte to form a plurality of detection complexes, wherein the two detection reagents comprise a first proximity probe and a second proximity probe, respectively;
• c. Contacting each detection complex formed in (b) with one or more connector oligonucleotides including a first connector probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second connector probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, the contacting step (c) is performed under conditions sufficient to ligate the first and second proximity probes to form a plurality of target sequences;
• d. Amplifying the plurality of target sequences to generate a plurality of amplicons each comprising a detection sequence and an anchoring sequence complement;
• e. Hybridizing each anchoring sequence to the anchoring sequence complement;
• f. Hybridizing a plurality of detection probes to the plurality of detection probe sequences;
• and
• g. Measuring the amount of analytes bound to the surface.

A method of detecting a plurality of analytes of interest in a sample comprising:

• a. Contacting the sample with a surface comprising a plurality of discrete binding domains, each binding domain comprising (i) a binding reagent for an analyte, and (ii) an anchoring reagent comprising an anchoring sequence complementary to an amplicon sequence, wherein the contacting step forms a plurality of surface-bound complexes;
• b. Contacting each of the surface-bound complexes with two detection reagents for the analyte to form a plurality of detection complexes, wherein the two detection reagents comprise a first proximity probe and a second proximity probe, respectively;
• c. Contacting each detection complex formed in (b) with one or more connector oligonucleotides including a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, the contacting step (c) is performed under conditions sufficient to form a plurality of circular DNA templates;
• d. Amplifying the plurality of circular DNA templates to generate a plurality of amplicons each comprising a plurality of detection sequences and an anchoring sequence complement;
• e. Hybridizing each anchoring sequence to the anchoring sequence complement;
• f. Hybridizing a plurality of detection probes to the plurality of detection probe sequences;
• and
• g. Measuring the amount of analytes bound to the surface.

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

• a. A surface comprising a binding reagent for the analyte and an anchoring reagent comprising an anchoring sequence complementary to 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 and a second proximity probe, respectively;
• ii. one or more connector oligonucleotides including a first connector probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second connector probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe;
• and
• iii. One or more detection probes complementary to the detection probe sequence.

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

• a. A surface comprising a binding reagent for the analyte and an anchoring reagent comprising an anchoring sequence complementary to 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 and a second proximity probe, respectively;
• ii. One or more connector oligonucleotides including a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe;
• and
• iii. One or more detection probes complementary to the detection probe sequence.

A kit for the measurement of a plurality of analytes of interest in a sample, the kit comprising:

• a. A surface comprising a plurality of discrete binding domains, each binding domain comprising (i) a binding reagent for an analyte and (ii) an anchoring reagent comprising an anchoring sequence complementary to an amplicon sequence;
• and
• b. In one or more containers, compartments, or vessels:
• i. Two detection reagents for each analyte, wherein the two detection reagents comprise a first proximity probe and a second proximity probe, respectively;
• iii. one or more connector oligonucleotides including a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe;
• and
• iii. One or more detection probes complementary to the detection probe sequence.