ACRIDINE LABELLED CONJUGATES AND PREPARATION METHODS THEREFOR AND CHEMILUMINESCENT KITS

• US 20190309030A1
• Filed: 07/05/2016
• Published: 10/10/2019
• Est. Priority Date: 07/05/2016
• Status: Abandoned Application

• Current Assignee: Shenzhen Yhlo Biotech Co., Ltd.
• Original Assignee: Shenzhen Yhlo Biotech Co., Ltd.
• Inventors: XIA, Fuzhen, LIU, Taoxu, ZHU, Liang, XIAO, Chengyong, QIAN, Chungen
• Application Number:
  US16/315,167
• Publication Number:
  US 20190309030A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 49/0026 Acridine dyes
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

Disclosed are an acridine marker conjugate and a preparation method therefor and a chemiluminescent kit. The acridine marker conjugate comprises an acridine substitute, a carrier protein and a protein to be marked sequentially connected. The carrier protein is a protein containing carboxyl and amino, a modified protein, a polypeptide or a modified polypeptide; and the protein to be marked is a protein containing amino, a modified protein, a polypeptide or a modified polypeptide.

Claims

1. An acridine-labelled conjugate, comprising:

a substituted acridine, a carrier protein, and a protein to be labelled which are sequentially linked;

the carrier protein is a protein, a modified protein, a polypeptide or a modified polypeptide containing a carboxyl group and an amino group, and the carrier protein reacts, by the amino group therein, with the substituted acridine to form a chemical bond;

the protein to be labelled is a protein, a modified protein, a polypeptide or a modified polypeptide containing an amino group, and the amino group in the protein to be labelled reacts with the carboxyl group in the carrier protein to form a —

NH—

CO—

structure, thereby linking the carrier protein and the protein to be labelled together.

2. The acridine-labelled conjugate according to claim 1, wherein the substituted acridine is acridinium ester, acridinic acid, acridine amide or acridine sulfonamide.

3. The acridine-labelled conjugate according to claim 1, wherein the carrier protein is bovine serum albumin, chicken serum albumin or hemocyanin.

4. The acridine-labelled conjugate according to claim 1, wherein the protein to be labelled is an antigen, a hapten or an antibody.

5. A method for preparing the acridine-labelled conjugate according to claim 1, comprising:

covalently cross-linking and fully reacting a substituted acridine with a carrier protein to obtain a substituted acridine-carrier protein conjugate, wherein the carrier protein is a protein, a modified protein, a polypeptide or a modified polypeptide containing a carboxyl group and an amino group, and the carrier protein reacts, by an amino group therein, with substituted acridine to form a chemical bond;

purifying the substituted acridine-carrier protein conjugate;

activating the carboxyl group in a purified substituted acridine-carrier protein conjugate by using a crosslinking agent; and

cross-linking and fully reacting a carboxyl group-activated substituted acridine-carrier protein conjugate with a protein to be labelled to obtain the acridine-labelled conjugate, wherein the acridine-labelled conjugate comprises a substituted acridine, a carrier protein and the protein to be labelled which are sequentially linked, the protein to be labelled is a protein, a modified protein, a polypeptide or a modified polypeptide containing an amino group, and the amino group in the protein to be labelled reacts with the carboxyl group in carrier protein to form a —

NH—

CO—

structure, thereby linking carrier protein and the protein to be labelled together.

6. The method according to claim 5, wherein a molar ratio of substituted acridine to the carrier protein ranges from 100:

1 to 20000;

1 in covalently crosslinking substituted acridine with the carrier protein.

7. The method according to claim 6, wherein a molar ratio of the carboxyl group-activated substituted acridine-carrier protein conjugate to the protein to be labelled ranges from 5:

1 to 1;

5 in cross-linking the carboxyl group-activated substituted acridine-carrier protein conjugate with a protein to be labelled.

8. The method according to claim 5, wherein the crosslinking agent comprises carbodiimide and hydroxysuccinimide in activating the carboxyl group in the purified substituted acridine-carrier protein conjugate by using the crosslinking agent.

9. The method according to claim 8, wherein carbodiimide is at least one selected from dicyclohexylcarbodiimide, 1-(3-dimethylamino propyl)-3-ethylcarbodiimide, and N,N′

-diisopropyl carbodiimide, and a molar ratio of carbodiimide to the substituted acridine-carrier protein conjugate ranges from 10;

1 to 5000;

1;

hydroxysuccinimide is at least one selected from N-hydroxysuccinimide and N-hydroxy sulfo succinimide, and a molar ratio of carbodiimide to hydroxysuccinimide ranges from 5;

1 to 1;

10.

10. A chemiluminescence kit for binding a protein to be labelled to form the acridine-labelled conjugate according to claim 1, comprising a substituted acridine and a carrier protein;

the carrier protein is a protein, a modified protein, a polypeptide or a modified polypeptide containing a carboxyl group and an amino group, and the carrier protein reacts, by the amino group therein, with the substituted acridine to form a chemical bond;

the protein to be labelled is a protein, a modified protein, a polypeptide or a modified polypeptide containing an amino group, and the amino group in the protein to be labelled reacts with the carboxyl group in the carrier protein to form a —

NH—

CO—

structure, thereby linking the carrier protein and the protein to be labelled together.