# US 2019/0317080 A1 · Kits for labeling of biomarkers and methods of using the same

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- Number: US 2019/0317080 A1
- Document ID: US20190317080A1
- Status: Published application
- Kind: A1
- Application number: 16/383,227
- Filed: 2019-04-12
- Published: 2019-10-17
- Earliest claimed: 2018-04-13
- Assignees: RareCyte, Inc.
- Inventors: Daniel E. CAMPTON; Yao SUN
- Web page: https://patentgrep.com/patent/US20190317080A1

## CPC

- G01N 33/526: {the element being adapted for a specific analyte}
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)}
- G01N 33/582: {with fluorescent label}
- G01N 33/588: {with semiconductor nanocrystal label, e.g. quantum dots}
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G01N 2800/60: Complex ways of combining multiple protein biomarkers for diagnosis
- G01N 2800/70: Mechanisms involved in disease identification (G01N2800/02 - G01N2800/44 take precedence)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Kits, compositions, and methods for labeling target materials or target analytes are considered. The composition can include one or more affinity molecules indirectly conjugated to one or more detection moieties. The kit can be used to detect a single biomarker or multiple biomarkers. The kit can be used to perform a single round of labeling or multiple rounds of labeling.

## Claims

### Claim 1

A kit comprising:
a first affinity molecule-first oligonucleotide conjugate;
a horseradish peroxidase (“HRP”) molecule-second oligonucleotide conjugate; and
a tyramide-first detection moiety conjugate,
wherein the first and second oligonucleotides are complementary.

### Claim 2 (depends on 1)

The kit of claim 1, wherein the tyramide and the first detection moiety are conjugated via complementary third and fourth oligonucleotides.

### Claim 3 (depends on 2)

The kit of claim 2, further comprising at least one signal disrupter to de-hybridize or cleave the third and fourth oligonucleotides.

### Claim 4 (depends on 1)

The kit of claim 1, wherein the tyramide and the first detection moiety are conjugated via a binding pair comprising first and second complementary molecules.

### Claim 5 (depends on 4)

The kit of claim 4, wherein the binding pair is a hapten-anti-hapten pair.

### Claim 6 (depends on 1)

The kit of claim 1, further comprising at least one signal disrupter to eliminate or reduce a signal capable of being provided by the first detection moiety.

### Claim 7 (depends on 1)

The kit of claim 1, wherein the first detection moiety is directly conjugated to the tyramide.

### Claim 8 (depends on 1)

The kit of claim 1, further comprising:
a second affinity molecule-third oligonucleotide conjugate;
a horseradish peroxidase (“HRP”) molecule-fourth oligonucleotide conjugate;
a tyramide-second detection moiety conjugate,
wherein the third and fourth oligonucleotides are complementary.

### Claim 9 (depends on 8)

The kit of claim 8, wherein the first and second affinity molecules are directed to different biomarkers.

### Claim 10 (depends on 8)

The kit of claim 8, wherein the first and second detection moieties have different emission wavelengths.

### Claim 11 (depends on 8)

The kit of claim 8, further comprising at least one signal disrupter to de-hybridize or cleave at least one of the first and second oligonucleotides or the third and fourth oligonucleotides.

### Claim 12 (depends on 8)

The kit of claim 8, further comprising at least one signal disrupter to eliminate or reduce a signal capable of being provided by at least one of the first and second detection moieties.

### Claim 13 (depends on 8)

The kit of claim 8, wherein the tyramide and the first detection moiety of the tyramide-first detection moiety conjugate are conjugated via complementary fifth and sixth oligonucleotides; and wherein the tyramide and the second detetion moiety of the tyramide-second detection moiety conjugate are conjugated via complementary seventh and eighth oligonucleotides.

### Claim 14 (depends on 13)

The kit of claim 13, further comprising at least one signal disrupter to de-hybridize or cleave one or more of the oligonucleotide pairs.

### Claim 15

A method for labeling a target analyte of a sample, the method comprising:
adding, to the sample, a first affinity molecule-first oligonucleotide conjugate;
adding, to the sample, a horseradish peroxidase (“HRP”) molecule-second oligonucleotide conjugate; and
adding, to the sample, a tyramide-first detection moiety conjugate,
wherein the first and second oligonucleotides are complementary and hybridize with each other.

### Claim 16 (depends on 15)

The method of claim 15, wherein the tyramide-first detection moiety conjugate is added after the first affinity molecule-first oligonucleotide conjugate and the HRP-second oligonucleotide.

### Claim 17 (depends on 15)

The method of claim 15, wherein the tyramide-first detection moiety conjugate is conjugated before the step of adding the tyramide-first detection moiety conjugate.

### Claim 18 (depends on 15)

The method of claim 15, wherein the step of adding the tyramide-first detection moiety conjugate further comprises the steps of:
adding, to the sample, a tyramide-third oligonucleotide conjugate; and
adding, to the sample, a fourth oligonucleotide-first detection moiety conjugate,
wherein the third and fourth oligonucleotides are complementary and hybiridize with each other.

### Claim 19 (depends on 15)

The method of claim 15, further comprising the steps of:
observing a first signal of the first detection moiety; and
modifying the first signal with a signal disrupter,
wherein the observing the first signal and modifying the first signal steps are performed after the adding steps.

### Claim 20 (depends on 19)

The method of claim 19, wherein the tyramide and the first detection moiety of the tyramide-first detection moiety conjugate are conjugated via complementary third and fourth oligonucleotides; and wherein the signal disrupter de-hybridizes the third and fourth oligonucleotides.

### Claim 21 (depends on 15)

The method of claim 15, wherein the tyramide and the first detection moiety are conjugated via a binding pair comprising first and second complementary molecules.

### Claim 22 (depends on 21)

The method of claim 21, wherein the binding pair is a non-oligonucleotide binding pair, an immune-type binding pair, a non-immune-type binding, or a hapten-anti-hapten pair.

### Claim 23 (depends on 15)

The method of claim 15, further comprising:
a second affinity molecule-third oligonucleotide conjugate;
a horseradish peroxidase (“HRP”) molecule-fourth oligonucleotide conjugate;
a tyramide-second detection moiety conjugate,
wherein the third and fourth oligonucleotides are complementary.

### Claim 24 (depends on 23)

The method of claim 23, wherein the first and second affinity molecules are directed to different biomarkers.

### Claim 25 (depends on 23)

The method of claim 23, wherein the first and second detection moieties have different emission wavelengths.

### Claim 26 (depends on 23)

The method of claim 23, further comprising the steps of:
observing a first signal of the first detection moiety; and
modifying the first signal with a signal disrupter.

### Claim 27 (depends on 26)

The method of claim 26, further comprising the step of observing a second signal of the second detection moiety, wherein the first and second signals have the same emission wavelength, and wherein the observing and modifying steps for the first signal of the first detection moiety are performed before adding the tyramide-second detection moiety conjugate.

### Claim 28 (depends on 23)

The method of claim 23, wherein the tyramide and the first detection moiety of the tyramide-first detection moiety conjugate are conjugated via complementary fifth and sixth oligonucleotides; and wherein the tyramide and the second detetion moiety of the tyramide-second detection moiety conjugate are conjugated via complementary seventh and eighth oligonucleotides.

### Claim 29 (depends on 15)

The method of claim 15, wherein the step of adding the tyramide-first detection moiety conjugate further comprises the steps of:
adding, to the sample, a tyramide-first complementary molecule conjugate; and
adding, to the sample, a second complementary molecule-first detection moiety conjugate,
wherein the first and second complementary molecules are complementary to each and form a binding pair.

### Claim 30 (depends on 29)

The method of claim 29, wherein the binding pair is a non-oligonucleotide binding pair, an immune-type binding pair, a non-immune-type binding, or a hapten-anti-hapten pair.