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US 11,697,840 B2Granted
Method of detecting analyte in a sample with binding reagent, first detection reagent, and second detection reagent
AssigneeMeso Scale Technologies, LLC.
InventorsEli N. Glezer, John Kenten
Life of this patent
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Abstract
The present invention is directed to improved methods for conducting immunoassays. The methods are designed to amplify signals in immunoassays and anchor immunoassay complexes employed therein.
Claims27
A method of detecting an analyte of interest in a sample comprising:
- a. contacting the sample with a surface comprising (i) a binding reagent for the analyte, and (ii) an anchoring reagent comprising an anchoring oligonucleotide, and binding the analyte to the binding reagent to form a surface-bound complex;
- b. contacting the surface-bound complex with a first detection reagent for the analyte comprising a first proximity probe and a second detection reagent for the analyte comprising a second proximity probe to form a detection complex comprising the binding reagent, the analyte, and the first and second detection reagents;
- c. contacting the detection complex formed in (b) with one or more connector oligonucleotides comprising:
- (i) a first connector probe complementary to a first region of the first proximity probe and a first region of the second proximity probe, and (ii) 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 ligating the first and second connector probes to form a target oligonucleotide;
- d. amplifying the target oligonucleotide to generate an amplicon comprising (i) a plurality of detection oligonucleotides and (ii) an anchoring oligonucleotide complement, wherein the anchoring oligonucleotide complement is complementary to the anchoring oligonucleotide;
- e. hybridizing the anchoring oligonucleotide to the anchoring oligonucleotide complement;
- f. hybridizing a plurality of detection probes to the plurality of detection oligonucleotides;
- and
- g. measuring the amount of detection probes bound to the surface, thereby detecting the analyte.
6 more elements
A method of detecting an analyte of interest in a sample comprising:
- a. contacting the sample with (i) a first detection reagent for the analyte that comprises a first proximity probe, and (ii) a second detection reagent for the analyte that comprises a second proximity probe to form a detection complex;
- b. contacting the detection complex formed in (a) with a surface comprising (i) a binding reagent for the analyte and (ii) an anchoring reagent comprising an anchoring oligonucleotide, thereby forming a surface-bound complex;
- c. contacting the surface-bound complex formed in (b) with one or more connector oligonucleotides comprising:
- (i) a first connector probe complementary to a first region of the first proximity probe and a first region of the second proximity probe, and (ii) 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 ligating the first and second connector probes to form a target oligonucleotide;
- d. amplifying the target oligonucleotide to generate an amplicon comprising (i) a plurality of detection oligonucleotides and (ii) an anchoring oligonucleotide complement, wherein the anchoring oligonucleotide complement is complementary to the anchoring oligonucleotide;
- e. hybridizing the anchoring oligonucleotide to the anchoring oligonucleotide complement;
- f. hybridizing a plurality of detection probes to the plurality of detection oligonucleotides;
- and
- g. measuring the amount of detection probes bound to the surface, thereby detecting the analyte.
6 more elements
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 of the plurality of analytes, and (ii) an anchoring reagent comprising an anchoring oligonucleotide, thereby forming a plurality of surface-bound complexes;
- b. contacting each surface-bound complex with:
- (i) a first detection reagent for the analyte of the surface-bound complex, wherein the first detection reagent comprises a first proximity probe, and (ii) a second detection reagent for the analyte of the surface-bound complex, wherein the second detection reagent comprises a second proximity probe, to form a plurality of detection complexes;
- c. contacting each detection complex formed in (b) with one or more connector oligonucleotides comprising:
- (i) a first connector probe complementary to a first region of the first proximity probe and a first region of the second proximity probe, and (ii) 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 ligating the first and second connector probes to form a target oligonucleotide;
- d. amplifying the target oligonucleotide of each detection complex to generate a plurality of amplicons, wherein each amplicon comprises (i) a plurality of detection oligonucleotides and (ii) an anchoring oligonucleotide complement, wherein the anchoring oligonucleotide complement is complementary to the anchoring oligonucleotide;
- e. hybridizing the anchoring oligonucleotide to the anchoring oligonucleotide complement;
- f. hybridizing a plurality of detection probes to the plurality of detection oligonucleotides;
- and
- g. measuring the amount of detection probes bound to the surface, thereby detecting the plurality of analytes.
7 more elements
Dependencies are inferred from the claim text.