# US 2021/0079445 A1 · Multi Component Detection

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- Number: US 2021/0079445 A1
- Document ID: US20210079445A1
- Status: Published application
- Kind: A1
- Application number: 16/800,394
- Filed: 2020-02-25
- Published: 2021-03-18
- Earliest claimed: 2011-07-28
- Assignees: Cell Signaling Technology, Inc.
- Applicants: Cell Signaling Technology, Inc.
- Inventors: Khanh Duc HUYNH; Wan Cheung CHEUNG; Roberto POLAKIEWICZ
- Web page: https://patentgrep.com/patent/US20210079445A1

## CPC

- C12Q 1/37: involving peptidase or proteinase
- C07K 16/18: against material from animals or humans
- C12N 9/52: derived from bacteria {or Archaea}
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 33/56966: {Animal cells}
- G01N 33/5759: {involving compounds localised on the membrane of tumour or cancer cells}
- C07K 2319/43: containing a FLAG-tag
- C07K 2319/61: containing an enzyme fusion for detection (lacZ, luciferase)
- C12Y 304/2207: Sortase A (3.4.22.70)
- G01N 2800/60: Complex ways of combining multiple protein biomarkers for diagnosis
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES

## Abstract

The disclosure provides methods for detecting the concurrent presence of at least two targets within a biological sample. The method includes contacting said biological sample with a first binding agent, said first binding agent operably linked to a first sortase molecule, wherein said first binding agent specifically binds to a first target; contacting said biological simple with a second binding agent, said second binding agent operably linked to a first sortase recognition sequence peptide, wherein said second binding agent specifically binds to a second target; adding a sortase substrate under conditions where a first sortase-mediated ligation of the sortase substrate to the first sortase recognition sequence will produce a ligation product, and detecting the ligation product, wherein detection of said ligation product indicates the concurrent presence of the first target and the second target in the biological sample. Also disclosed are kits comprising reagents for performing the methods as claimed.

## Claims

### Claim 1

A method for detecting the concurrent presence of at least two targets within a biological sample, comprising:
(a) contacting said biological sample with a first binding agent, said first binding agent operably linked to a first sortase molecule, wherein said first binding agent specifically binds to a first target;
(b) contacting said biological sample with a second binding agent, said second binding agent operably linked to a first sortase recognition sequence peptide, wherein said second binding agent specifically binds to a second target;
(c) adding a sortase substrate under conditions where a first sortase-mediated ligation of the sortase substrate to the first sortase recognition sequence will produce a ligation product, and
(d) detecting the ligation product, wherein detection of said ligation product indicates the concurrent presence of the first target and the second target in the biological sample.

### Claim 2

(canceled)

### Claim 3 (depends on 1)

The method of claim 1, wherein the first sortase molecule is a Sortase A molecule.

### Claim 4 (depends on 3)

The method of claim 3, wherein the Sortase A molecule is from Staphylococcus aureus.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first sortase recognition sequence peptide comprises the amino acid sequence LPXTG, where X is any amino acid residue (SEQ ID NO: 1).

### Claim at position 6

-7. (canceled)

### Claim 8 (depends on 1)

The method of claim 1, wherein the sortase substrate comprises the amino acid sequence GGG.

### Claim 9 (depends on 1)

The method of claim 1, wherein detecting the ligation product performed using a fourth binding agent that specifically binds to the ligation product.

### Claim 10 (depends on 9)

The method of claim 9, wherein the fourth binding agent is detectable.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first sortase molecule is directly attached to the first binding agent.

### Claim 12 (depends on 1)

The method of claim 1, wherein the first sortase molecule is indirectly attached to the first binding agent.

### Claim 13 (depends on 1)

The method of claim 1, wherein the first sortase recognition sequence peptide is directly attached to the second binding agent.

### Claim 14 (depends on 1)

The method of claim 1, wherein the first sortase recognition sequence peptide is indirectly attached to the second binding agent.

### Claim at position 14

-16. (canceled)

### Claim 17 (depends on 1)

The method of claim 1, wherein the first target and the second target are on the same molecule.

### Claim at position 16

-19. (canceled)

### Claim 20 (depends on 1)

The method of claim 1, wherein the first binding agent is an antibody.

### Claim 21 (depends on 1)

The method of claim 1, wherein the second binding agent is an antibody.

### Claim 22

(canceled)

### Claim 23 (depends on 9)

The method of claim 9, wherein the fourth binding agent is an antibody.

### Claim 24 (depends on 1)

The method of claim 1, wherein the biological sample is selected from the group consisting of a cell, a biopsy sample, a blood sample, a tissue sample, a saliva sample, a tear sample, a semen sample, cerebrospinal fluid sample, a bone marrow sample, a bone marrow sample, and a circulating tumor cell sample.

### Claim 25 (depends on 1)

The method of claim 1, wherein the biological sample is from a human.

### Claim 26

A kit comprising a first sortase recognition sequence directly attached to a first member of a first binding member pair, a first sortase molecule directly attached to a first member of a second binding member pair; a sortase substrate; and instructions for using the kit to detect the concurrent presence of at least two targets within a biological sample.

### Claim 27 (depends on 26)

The kit of claim 26, wherein the sortase substrate is directly attached to a first member of a third binding member pair.

### Claim at position 25

-31. (canceled)