# US 8,465,938 B2 · Method for producing complex of biotin-labeled Cypridina (Cypridina noctiluca) luciferase with streptoavidin and method for stabilizing the same

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- Number: US 8,465,938 B2
- Document ID: US8465938B2
- Status: Granted
- Kind: B2
- Application number: 12/959,729
- Filed: 2010-12-03
- Published: 2012-03-29
- Granted: 2013-06-18
- Earliest claimed: 2006-07-24
- Assignees: National Institute of Advanced Industrial Science and Technolgy
- Applicants: Yoshihiro Ohmiya; Chun Wu; Satoru Ohgiya; Kosei Kawasaki
- Inventors: Yoshihiro Ohmiya; Chun Wu; Satoru Ohgiya; Kosei Kawasaki
- Web page: https://patentgrep.com/patent/US8465938B2

## CPC

- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12Q 1/66: involving luciferase
- G01N 33/532: Production of labelled immunochemicals
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)

## Abstract

The present invention relates to a method for producing Cypridina luciferase labeled with hydrophilic biotin, characterized in that a biotin reagent containing a polyalkylene glycol structure as a spacer is reacted with Cypridina luciferase, and biotin-labeled Cypridina luciferase wherein a sugar chain in Cypridina luciferase has been biotinylated.

## Claims

### Claim 1

An immunoassay method comprising steps of:
reacting an analyte with a first complex comprising a biotin-labeled Cypridina luciferase, a polyvalent avidin substance and an antibody capable of recognizing the analyte, to form a second complex, and
contacting a luciferin with the second complex and measuring a luminescence, wherein:
a biotin of the biotin-labeled Cypridina luciferase
is bound at a position corresponding to a lysine residue K180 and/or K203 of SEQ ID NO: 1 via a polyethylene glycol moiety represented by formula —(CH2CH2O)n—
wherein n is an integer of 2 to 50, or
is bound via a sugar chain with a hydrazide linkage;
the biotin-labeled Cypridina luciferase is labeled with 2 to 5 biotins; and
the polyvalent avidin substance is selected from the group consisting of avidin, streptavidin and NeutrAvidin.

### Claim 2 (depends on 1)

The immunoassay method according to claim 1, wherein the immunoassay method is selected from the group consisting of a direct immunoassay method, an indirect immunoassay method, a sandwich ELISA method and an ELI SPOT method.

### Claim 3 (depends on 1)

The immunoassay method according to claim 1, wherein the analyte is a protein or DNA.