Acridine marker conjugate, preparation method of acridine marker conjugate and chemical luminescent kit

A conjugate and acridine technology, applied in the field of chemiluminescent kits, acridine-labeled conjugates and their preparation, can solve the problems of reduced activity of acridine-labeled conjugates and affecting the sensitivity of immunoassays, etc.

Inactive Publication Date: 2016-11-16
SHENZHEN YHLO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, in the acridine-labeled conjugates prepared by traditional methods, the acridine substituent is combined with the protein to be labeled through carbodiimide, and the acridine substituent often interferes with the active site on the protein to be labeled, resulting in acridine labeling. The activity of the conjugate is reduced, affecting the sensitivity of the immunoassay

Method used

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  • Acridine marker conjugate, preparation method of acridine marker conjugate and chemical luminescent kit
  • Acridine marker conjugate, preparation method of acridine marker conjugate and chemical luminescent kit
  • Acridine marker conjugate, preparation method of acridine marker conjugate and chemical luminescent kit

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preparation example Construction

[0047] Such as figure 1 The preparation method of the above-mentioned acridine-labeled conjugate shown includes the following steps:

[0048] S10, covalently cross-linking the acridine substituent with the carrier protein, and obtaining the acridine substituent-carrier protein conjugate after sufficient reaction.

[0049] In the operation of covalently cross-linking the acridine substituent and the carrier protein, the molar ratio of the acridine substituent to the carrier protein is 100-20000:1.

[0050] Preferably, the molar ratio of the acridine substituent to the carrier protein is 500-5000:1.

[0051] The carrier protein is a protein, modified protein, polypeptide or modified polypeptide containing carboxyl and amino groups, and the carrier protein is connected by a chemical bond through the reaction of the amino group on the carrier protein with the acridine substituent.

[0052] The carrier protein can be a protein or polypeptide that itself has carboxyl and amino gro...

Embodiment 1

[0098] Dissolve 1 mg BSA in 1 mL of 150 mM PBS buffer (pH 7.4), add 40 μL of acridinium ester (10 mg / mL dissolved in DMF) and react at 25°C for 4 h, use 5 mL of 7KD molecular weight cut-off desalting column (Thermofish Company) to dilute with 150 mM PBS ( pH 7.4) buffer solution was used as the replacement buffer solution, and passed through the column for 3 times to remove free acridinium esters and reaction by-products to obtain an acridinium-BSA solution.

[0099] EDC (final concentration: 10 mmol / L) and NHS (final concentration: 20 mmol / L) were added to the above-mentioned purified acridine-BSA solution. After reacting at 25°C for 10 minutes, TSH antibody (manufacturer: Santa Cruz biotechnology, product number: sc-418393) was added, mixed evenly, and placed at 25°C for 4 hours, and 5 mL of a 7KD molecular weight cut-off desalting column (Thermofish Company) was used to dilute with 150 mM PBS ( pH 7.4) buffer was used as the replacement buffer, and the column was passed 3 t...

Embodiment 2

[0101] Dissolve 1mg BSA in 1mL 150mM PBS buffer (pH 7.4), add 40μL acridinium ester (10mg / mL dissolved in DMF) and react at 25°C for 4h, use 5mL 7KD molecular weight cut-off desalting column (Thermofish Company) (pH 7.4) buffer solution was used as the replacement buffer solution, and the column was passed 3 times to remove free acridinium esters and reaction by-products to obtain an acridinium-BSA solution.

[0102] EDC (final concentration: 10 mmol / L) and NHS (final concentration: 20 mmol / L) were added to the above-mentioned purified acridine-BSA solution. After reacting at 25°C for 10 minutes, add estradiol antigen (manufacturer: abcam, product number: ab120657), mix well, place at 25°C for 4 hours, and use 5mL 7KD molecular weight cut-off desalting column (Thermofish Company) to dissolve 150mM PBS (pH 7.4 ) buffer as a liquid replacement buffer, passed through the column 3 times to remove free EDC, NHS and reaction by-products to obtain an acridinium ester-labeled estradio...

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Abstract

The invention discloses an acridine-labeled conjugate, a preparation method thereof, and a chemiluminescence kit. The acridine-labeled conjugate includes acridine substituents connected in sequence, a carrier protein and a protein to be labeled; the carrier protein is a carboxyl- and amino-containing Protein, modified protein, polypeptide or modified polypeptide; the protein to be labeled is amino-containing protein, modified protein, polypeptide or modified polypeptide. The carrier protein of this acridine-labeled conjugate forms a chemical bond connection through the reaction of the amino group on the carrier protein with the acridine substituent, and the amino group on the protein to be labeled reacts with the carboxyl group on the carrier protein to form a ‑NH‑CO‑structure so that the carrier protein Linked with the protein to be labeled, the binding site is relatively definite, avoiding the interference of the acridine substitute on the active site on the protein to be labeled, and the activity of this acridine-labeled conjugate is relatively high.

Description

technical field [0001] The invention relates to the field of in vitro detection, in particular to an acridine-labeled conjugate, a preparation method thereof, and a chemiluminescence kit. Background technique [0002] Chemiluminescence labeling immunoassay, also known as chemiluminescence immunoassay (CLIA), is an immunoassay method that uses chemiluminescent agents to directly label antigens, haptens or antibodies. The chemiluminescent substances used for labeling include acridinium substituents. According to different substituents, acridinium substituents are divided into two categories: acridinium ester (acridinium ester, AE) and acridinium sulfonamide, both of which are effective luminescent label, by initiating luminescent reagents (NaOH, H 2 o 2 ) to emit light by action, the intense direct luminescence is completed within a second, and the luminescence is rapid flickering. [0003] Acridine substitutes are used as chemiluminescent markers for immunoassays. The chem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/76C07K19/00C07K1/13C07K1/08
CPCG01N33/68C07K14/00C07K14/765C07K14/795C07K16/26C07K2319/00G01N21/76G01N33/6854G01N2458/00
Inventor 夏福臻刘陶旭祝亮肖成勇钱纯亘
Owner SHENZHEN YHLO BIOTECH
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