Cup type time-resolved fluorescence BNP analysis method and reagent kit based on microspheres

A technology of time-resolved fluorescence and analysis method, which is applied in the field of cup-type time-resolved fluorescence BNP analysis method and kit based on microspheres, which can solve the problems of unsatisfactory detection precision, complicated detection steps, poor precision, etc., and reduce space Effects of steric hindrance, shorter detection time, and higher detection sensitivity

Inactive Publication Date: 2016-08-24
SHANGHAI UPPER BIO TECH PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The purpose of the present invention is to overcome the shortcomings of the above-mentioned existing fluorescent immunochromatographic method for measuring BNP detection precision and the time-resolved fluorescence detection method needs dissociation enhancement process, complicated detection steps, long time, poor precision, etc., and provides a micro-based Ball cup time-resolved fluorescent BNP analysis method and kit

Method used

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  • Cup type time-resolved fluorescence BNP analysis method and reagent kit based on microspheres
  • Cup type time-resolved fluorescence BNP analysis method and reagent kit based on microspheres
  • Cup type time-resolved fluorescence BNP analysis method and reagent kit based on microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Preparation of detection cuvette

[0059] (1) Detection cuvette coating

[0060] The BNP antibody 50E1 (Hytest Company) was diluted to 20 μg / ml with 0.2M phosphate buffer (pH7.8), 200 μL / well, coated at 37° C. for 4 hours, and washed.

[0061] (2) Detect cuvette closure

[0062] Add 300 μL / well of blocking buffer to the reaction cup, block at 37°C for 4 hours, discard the blocking solution in the coated reaction cup, and pat dry.

[0063] (3) Detection of cuvette drying

[0064] The sealed cuvette was placed in a 37° C. drying oven with a humidity lower than 30% for 8 hours, sealed and dried for storage.

[0065] 2. Preparation of fluorescently labeled antibodies

[0066] (1) Pretreatment of fluorescent particles

[0067] Take 1mg of carboxyl time-resolved fluorescent microspheres (300nm, 0.1ml, 10mg / mL, Bangslab Company), add 60ul of 500mM MES (2-(N-morpholine)ethanesulfonic acid) buffer, pH 6.0, add 0.2mg of 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydr...

Embodiment 2

[0095] 1. Preparation of detection cuvette

[0096] (1) Detection cuvette coating

[0097] The BNP antibody 50E1 (Hytest Company) was diluted to 20 μg / ml with 0.2M phosphate buffer (pH7.8), 200 μL / well, coated at 37° C. for 4 hours, and washed.

[0098] (2) Detect cuvette closure

[0099] Add 300 μL / well of blocking buffer to the reaction cup, block at 37°C for 4 hours, discard the blocking solution in the coated reaction cup, and pat dry.

[0100] (3) Detection of cuvette drying

[0101] The sealed cuvette was placed in a 37° C. drying oven with a humidity lower than 30% for 8 hours, sealed and dried for storage.

[0102] 2. Preparation of fluorescently labeled antibodies

[0103] (1) Pretreatment of fluorescent particles

[0104] Take 1mg of carboxyl time-resolved fluorescent microspheres (300nm, 0.1ml, 10mg / mL, Bangslab Company), add 60ul of 500mM MES (2-(N-morpholine)ethanesulfonic acid) buffer, pH 6.0, add 0.2mg of 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydr...

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Abstract

The invention discloses a cup type time-resolved fluorescence BNP analysis method and a reagent kit based on microspheres. The method includes the steps that the inner surface of a detection reaction cup is coated with BNP monoclonal antibodies or polyclonal antibodies; the time-resolved fluorescence microspheres and the BNP monoclonal antibodies or polyclonal antibodies are subjected to covalent bond cross-linking to prepare fluorescence-labeled antibodies; a BNP sample to be detected is added into the coated detection reaction cup, then the fluorescence-labeled antibodies are added, and incubation is carried out at 25-40 DEG C; cleaning is carried out to remove unbound antigens and fluorescence-labeled antibodies, and fluorescence signals in the detection reaction cup are tested under the excitation of 340-380 nm exciting light; the fluorescence signals are compared with a standard curve, and the BNP concentration of the BNP sample to be detected is obtained. By means of the method, detection can be completed within a short detection time, the detection time is shortened, and the detection sensitivity is effectively improved.

Description

technical field [0001] The invention relates to the technical field of BNP detection, in particular to a microsphere-based cup-type time-resolved fluorescent BNP analysis method and a kit. Background technique [0002] Heart failure is a clinical syndrome of signs and symptoms due to cardiac dysfunction. Although some current drugs such as angiotensin-converting enzyme (ACE) inhibitors and β-receptor blockers can significantly improve the quality of life of patients and delay the deterioration of the disease, heart failure cannot be cured unless heart transplantation is performed. of. Early diagnosis of heart failure and avoiding its progression are critical issues in healthcare today, and the sooner treatment is started, the better for the patient. [0003] However, heart failure (especially in its early stages) can be difficult to diagnose. The typical symptoms of heart failure, such as shortness of breath, ankle swelling, fatigue, etc., are not specific, and some patie...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/325
Inventor 石晓强李福刚徐建新周奕璇杨晶
Owner SHANGHAI UPPER BIO TECH PHARMA
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