Acridine marker conjugate and preparation method thereof and chemiluminescent kit

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

Inactive Publication Date: 2016-10-26
SHENZHEN YHLO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 and preparation method thereof and chemiluminescent kit
  • Acridine marker conjugate and preparation method thereof and chemiluminescent kit
  • Acridine marker conjugate and preparation method thereof and chemiluminescent kit

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preparation example Construction

[0045] Such as figure 1 The preparation method of the above-mentioned acridine-labeled conjugate shown includes the following steps:

[0046] S10, using the activated acridine substituent to covalently cross-link the polyamino acid, and obtain the acridine substituent-polyamino acid conjugate after sufficient reaction.

[0047] In the operation of using the activated acridine substituent and the polyamino acid for covalent crosslinking, the molar ratio of the activated acridine substituent to the polyamino acid is 1-10000:1.

[0048] Preferably, the molar ratio of the acridine substituent to the polyamino acid is 10-200:1.

[0049] The activated acridine substituent may be a carboxyl activated acridine substituent. The activated acridine substituent can be activated by carbodiimide and hydroxysuccinimide. The specific activation operation is as follows: add acridine substituent in the buffer solution, after fully dissolved, add EDC and NHS, at 25°C After reacting for 10 min...

Embodiment 1

[0101] Dissolve poly-lysine in 1 mL of 150 mM PBS buffer (pH 7.4), with a final concentration of 10 nmol / L, add 10 μL of 10 mmol / L acridinium ester dissolved in DMSO solvent, react at 25 °C for 1 h, and use 5 mL of 7KD molecular weight cut-off The desalting column (Thermo fish company) used 150mM PBS (pH7.4) buffer as the liquid exchange buffer, and passed through the column 3 times to remove free acridinium esters and reaction by-products to obtain an acridine-polylysine solution.

[0102] EDC (final concentration: 5 mmol / L) and NHS (final concentration: 10 mmol / L) were added to the above-mentioned purified acridine-polylysine solution. After reacting at 25°C for 30 minutes, add mercaptoethanol with a final concentration of 10 mM to obtain activated acridine-polylysine;

[0103] After adding 1mg of anti-PTH monoclonal antibody (manufacturer: Abnova, product number: PAB5103, 6.67nmol), mix well, place it at 25°C for 1h, and use 5mL 7KD molecular weight cut-off desalting column...

Embodiment 2

[0105] Dissolve poly-lysine in 1 mL of 150 mM PBS buffer (pH 7.4), with a final concentration of 10 nmol / L, add 10 μL of 10 mmol / L acridinium ester dissolved in DMSO solvent, react at 25 °C for 1 h, and use 5 mL of 7KD molecular weight cut-off The desalting column (Thermo fish company) used 150mM PBS (pH7.4) buffer as the liquid exchange buffer, and passed through the column 3 times to remove free acridinium esters and reaction by-products to obtain an acridine-polylysine solution.

[0106] EDC (final concentration: 5 mmol / L) and NHS (final concentration: 10 mmol / L) were added to the above-mentioned purified acridine-polylysine solution. After reacting at 25°C for 30 minutes, use a 5mL 7KD molecular weight cut-off desalting column (Thermo fish company) with 150mM PBS (pH 7.4) buffer as the replacement buffer, and pass through the column 3 times to remove free acridinium esters and reaction by-products. Product, obtain acridine-polylysine solution. After adding 1mg of anti-PTH...

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Abstract

The invention discloses an acridine marker conjugate and a preparation method thereof and a chemiluminescent kit. The acridine marker conjugate comprises an acridine substitute, polyamino acid and to-be-marked protein; the polyamino acid contains carboxyl and amino; the to-be-marked protein is amino protein, modified protein, polypeptide or modified polypeptide. The polyamino acid of the acridine marker conjugate reacts with the acridine substitute through the amino of the polyamino acid to form chemical bond connection, the amino of the to-be-marked protein reacts with the carboxyl of the polyamino acid to form a -NH-CO- structure, and accordingly the polyamino acid and the to-be-marked protein are connected together, active sites of the to-be-marked protein are protected from being interfered by the acridine substitute due to relative determinacy of binding sites, and the acridine marker conjugate is relatively high in activity.

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): G01N21/76C07K19/00C07K1/13C07K1/08
CPCC07K14/00C07K14/765C07K14/795C07K16/26C07K2319/00G01N21/76
Inventor 钱纯亘肖成勇祝亮刘陶旭夏福臻
Owner SHENZHEN YHLO BIOTECH
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