Acridine marker conjugate and preparation method thereof and chemiluminescence immunoassay kit

A technology of conjugates and markers, which can be used in peptide preparation methods, chemiluminescence/bioluminescence, biological testing, etc., and can solve the problems of decreased activity of acridine-labeled conjugates and affecting the sensitivity of immunoassays, etc.

Active Publication Date: 2016-11-23
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, since the acridine substituent and the substance to be labeled need to be directly combined through carbodiimide, the acridine substituent often interferes with the active site on the protein to be labeled. There are few active sites on the marker that can be selected for binding, resulting in reduced activity of acridine-labeled conjugates, which affects the sensitivity of immunoassays

Method used

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  • Acridine marker conjugate and preparation method thereof and chemiluminescence immunoassay kit
  • Acridine marker conjugate and preparation method thereof and chemiluminescence immunoassay kit
  • Acridine marker conjugate and preparation method thereof and chemiluminescence immunoassay kit

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preparation example Construction

[0050] Such as figure 1 The above-mentioned preparation method of the acridine-labeled conjugate includes the following steps S110-S120.

[0051] S110, using the activated acridine substituent to react with the linking carrier to obtain an intermediate product B.

[0052] Among them, the intermediate product B is an acridine substituent-linking carrier combination, and the linking carrier contains amino and carboxyl groups or the connecting carrier contains amino and disulfide bonds. Further, the connecting carrier can be a connection containing amino, carboxyl and disulfide bonds. The carrier, the acridine substituent reacts with the amino group on the linking carrier to form a -CO-NH- structure so as to connect the acridine substituent to the linking carrier.

[0053] Specifically, the acridine substituent is acridine ester, acridine acid, acridine amide or acridine sulfonamide. Specifically, it can be AE-NHS (10-methyl-acridine-9-N-succinimide ester formate), DMAE-NHS (2'...

Embodiment 1

[0121] Dissolve KLH in 1 mL of 150 mM PBS buffer (pH 7.4) to a final concentration of 20 nmol / L. Add 10 μL of 10 mmol / L acridinium ester dissolved in DMSO solvent. React at 25°C for 1 h, use 5 mL of 7KD molecular weight cut-off desalting column (Thermofish Company) with 150 mM PBS (pH 7.4) buffer as the liquid exchange buffer, and pass through the column for 3 times to remove free acridinium esters and reaction by-products to obtain Acridine ester-KLH solution.

[0122] Dissolve 1 mg of thyroglobulin (manufacturer: biospacific, product number: J19400, 1.5 nmol) in 1 mL of 50 mMMES buffer (pH 4.5), add EDC (final concentration: 5 mmol / L) and EMCH (final concentration: 10 mmol / L), After reacting at 25°C for 30 minutes, use 5mL 7KD molecular weight cut-off desalting column (Thermo fish company) with 150mM PBS (pH7.4) buffer as the replacement buffer, and pass through the column 3 times to remove free EDC, EMCH and By-product, the activated thyroglobulin-EMCH is obtained.

[01...

Embodiment 2

[0126] Dissolve KLH in 1 mL of 150 mM PBS buffer (pH 7.4) to a final concentration of 20 nmol / L, and add 10 μL of 10 mmol / L acridinium ester dissolved in DMSO solvent. React at 25°C for 1 h, use 5mL 7KD molecular weight cut-off desalting column (Thermofish Company) with 50mM MES (pH 4.5) buffer as the replacement buffer, pass through the column for 3 times, remove free acridinium esters and reaction by-products, and obtain Acridine ester-KLH solution.

[0127] EDC (final concentration is 5mmol / L) and EMCH (final concentration is 10mmol / L) are added in the acridinium ester-KLH, after reacting for 30 minutes at 25 ℃, use the desalting column (Thermo fish company) of 5mL 7KD molecular weight cut-off to 150mM PBS (pH 7.4) buffer was used as the replacement buffer, and the column was passed 3 times to remove free EDC, EMCH and by-products to obtain activated acridinium ester-KLH-EMCH;

[0128] Dissolve 1mg of anti-procalcitonin mouse monoclonal antibody antibody (manufacturer: Hyt...

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Abstract

The invention discloses an acridine marker conjugate and a preparation method thereof and a chemiluminescence immunoassay kit. The acridine marker conjugate comprises an acridine substitute, a connecting carrier, a difunctional cross-linking agent and an object to be marked which are connected in sequence. The acridine substitute and amidogen on the connecting carrier react to form a -CO-NH- structure so as to connect the acridine substitute with the connecting carrier. By means of the connecting carrier and the difunctional cross-linking agent containing a maleimide group and a hydrazide group, the acridine substitute and the object to be marked are not subjected to chemical bond connection directly, interference is formed by the acridine substitute on an active site on the object to be marked is avoided, the activity of the acridine marker conjugate is made higher, and the sensitivity of immunoassay is improved.

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 immunoassay kit. Background technique [0002] Chemiluminescence labeling immunoassay, also known as chemiluminescence immunoassay (CLIA), is an immunoassay method in which antigens, haptens, antibodies or carbohydrates are directly labeled with chemiluminescent agents. Chemiluminescent substances commonly used for labeling include acridinium esters (AE). It starts the luminescent reagent (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 compounds are used as markers for immunoassays. Their chemical reactions are simple, fast, and do not require catalysts. The competition method is used to detect small molecule antigens, and the sandwich method is used to detect macromolecular ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/13C07K1/08G01N21/76G01N33/68
CPCC07K14/00C07K14/795C07K16/18C07K2319/00G01N21/76G01N33/6803G01N2800/046G01N2800/7095
Inventor 钱纯亘刘陶旭肖成勇祝亮夏福臻
Owner SHENZHEN YHLO BIOTECH
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