# US 9,040,252 B2 · Method for increasing and regulating light emission from a chemiluminescent reaction

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,040,252 B2
- Document ID: US9040252B2
- Status: Granted
- Kind: B2
- Application number: 13/167,137
- Filed: 2011-06-23
- Published: 2012-01-12
- Granted: 2015-05-26
- Earliest claimed: 2010-07-06
- Assignees: CYANAGEN S.r.l.
- Applicants: Leopoldo Della Ciana; Federica Rodeghiero; Rossana Perciaccante
- Inventors: Leopoldo Della Ciana; Federica Rodeghiero; Rossana Perciaccante
- Web page: https://patentgrep.com/patent/US9040252B2

## CPC

- G01N 33/582: {with fluorescent label}
- C12Q 1/28: involving peroxidase
- G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding}
- 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

Method for increasing and regulating the emission of light from a chemiluminescent reaction including luminol, a peroxidase enzyme, an oxidant and an electron mediator (primary enhancer) through the use of an acylation catalyst (secondary enhancer) belonging to the class of N-azoles, i.e., a class of five-membered nitrogen heteroaromatic ring compounds containing at least one other atom of nitrogen. N-azoles, which are especially useful as secondary enhancers are imidazole, 1-methylimidazole, 1,2,3-triazole and 1,2,4-triazole. The invention also describes the use in diagnostic assays of chemiluminescent substrates containing said N-azoles, as secondary enhancers.

## Claims

### Claim 1

A method for increasing the light emission produced by the chemiluminescent reaction of luminol, a peroxidase enzyme, an oxidant and a primary enhancer, wherein said primary enhancer is sodium 3-(phenothiazin-10-yl)propane-1-sulfonate, and wherein said chemiluminescent reaction occurs in the presence of a secondary enhancer wherein said secondary enhancer is selected from the group consisting of imidazole, 1-methylimidazole, 1,2,3-triazole, and 1,2,4-triazole.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said peroxidase enzyme is selected from the group consisting of horseradish peroxidase, soybean peroxidase, and sweet potato peroxidase.

### Claim 3 (depends on 1)

The method according to claim 1, wherein said peroxidase is free or conjugated to a ligand.

### Claim 4 (depends on 1)

The method according to claim 1, wherein said oxidant is sodium perborate or hydrogen peroxide.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said chemiluminescent reaction is carried out at a off between 8.0 and 10.0.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said chemiluminescent reaction is carried out at a pH between 8.3 to 9.3.

### Claim 7 (depends on 1)

A method for performing a blot assay of an analyte in a sample, wherein said method employs a method for increasing the light emission produced by the chemiluminescent reaction of luminol according to claim 1 as means of detection and quantification of said analyte.

### Claim 8 (depends on 1)

A method for performing an ELISA assay of an analyte in a sample, wherein said method employs a method for increasing the light emission produced by the chemiluminescent reaction of luminol according to claim 1 as means of detection and quantification of said analyte.

### Claim 9

A kit for performing an assay for determining an analyte in a sample, wherein the kit comprises luminol, an oxidant, a primary enhancer in a form of an electron mediator, wherein said primary enhancer is sodium 3-(phenothiazin-10-yl)propane-1-sulfonate, and a secondary enhancer is selected from the group consisting of imidazole, 1-methylimidazole, 1,2,3-triazole, and 1,2,4-triazole.

### Claim 10 (depends on 9)

The kit according to claim 9, wherein luminol is present in a first vial and the oxidant is present in a second vial, and wherein the primary enhancer and the secondary enhancer are present either in the first vial or in the second vial or in both vials.

### Claim 11 (depends on 9)

The kit according to claim 9, wherein the kit further comprises a peroxidase enzyme.

### Claim 12 (depends on 9)

The kit according to claim 9, wherein luminol or the peroxidase enzyme are conjugated or are suitable to be conjugated to a ligand able to bind the analyte.

### Claim 13 (depends on 11)

The kit according to claim 11, wherein said peroxidase enzyme is selected from the group consisting of horseradish peroxidase, soybean peroxidase, and sweet potato peroxidase.

### Claim 14 (depends on 9)

The kit according to claim 9, wherein said oxidant is sodium perborate or hydrogen peroxide.