Assay methods

• US 10,114,015 B2
• Filed: 03/13/2014
• Issued: 10/30/2018
• Est. Priority Date: 03/13/2013
• Status: Active Grant

• Current Assignee: Meso Scale Technologies, LLC
• Original Assignee: Meso Scale Technologies, LLC
• Inventors: Glezer, Eli N., Kenten, John
• Primary Examiner(s): Crawford, Erik B
• Application Number:
  US14/208,040
• Publication Number:
  US 20140274775A1
• Time in Patent Office: 1,692 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2565/401 Signal amplification by chemical polymerisation
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/549 characterised by the capture oligonucleotide being a reporter labelled capture oligonucleotide
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms

Abstract

The present invention is directed to methods for reducing cross-reactivity between species employed in multiplexed immunoassays.

Claims

1. 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.

2. The kit of claim 1 further comprising (b)(iv) DNA polymerase.

3. The kit of claim 1 wherein the detectable label is an ECL label and the kit further comprises (b)(v) an ECL co-reactant.

4. The kit of claim 1 wherein said one or more connector oligonucleotides is one connector oligonucleotide that comprises said first connector probe sequence and said second connector probe sequence.

5. The kit of claim 1 wherein said one or more connector oligonucleotides is a first and a second connector oligonucleotide, wherein said first connector oligonucleotide comprises said first connector probe sequence and said second connector oligonucleotide comprises said second connector probe sequence.

6. The kit of claim 1 wherein the one or more detection probes further comprises an enzyme or a chemically reactive species that has a chemical activity having a measurable light scattering signal, absorbance signal or fluorescence signal.

7. The kit of claim 1 wherein the binding domain comprises a first binding domain and a second binding domain, wherein the first binding domain and the second binding domain are adjacent each other, and wherein the binding reagent is bound to the first binding domain and the anchoring reagent is bound to the second binding domain.

8. The kit of claim 1, wherein the binding reagent and the anchoring reagent are both bound to the same binding domain.