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Cup-type time-resolved fluorescent analysis method and kit for NT-proBNP based on microspheres

A time-resolved fluorescence and analysis method technology, which is applied in the field of cup-type time-resolved fluorescence NT-proBNP analysis methods and kits, can solve the problems of unsatisfactory detection precision, complicated detection steps, poor precision, etc., and achieves reduction of steric hindrance. effect, shorten the detection time, and improve the detection sensitivity

Inactive Publication Date: 2016-08-31
SHANGHAI UPPER BIO TECH PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The object of the present invention is to overcome the disadvantages of the above-mentioned existing fluorescent immunochromatography method for measuring NT-proBNP detection precision is not ideal and time-resolved fluorescence detection method needs dissociation enhancement process, detection steps are complicated, time is long, and precision is poor, etc., to provide a Cup type time-resolved fluorescent NT-proBNP analysis method and kit based on microspheres

Method used

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  • Cup-type time-resolved fluorescent analysis method and kit for NT-proBNP based on microspheres
  • Cup-type time-resolved fluorescent analysis method and kit for NT-proBNP based on microspheres
  • Cup-type time-resolved fluorescent analysis method and kit for NT-proBNP based on microspheres

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Experimental program
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Effect test

Embodiment 1

[0058] 1. Preparation of detection cuvette

[0059] (1) Detection cuvette coating

[0060] NT-proBNP antibody 15C4 (Hytest Company) was diluted to 20 μg / ml with 0.2M phosphate buffer (pH 7.8), 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 (200nm, 0.1ml, 10mg / mL, Bangslab company), add 60ul 500mM MES (2-(N-morpholine)ethanesulfonic acid) buffer, pH 6.0, add 0.2mg 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydroch...

Embodiment 2

[0094] (1) Detection cuvette coating

[0095] NT-proBNP antibody 15C4 (Hytest Company) was diluted to 20 μg / ml with 0.2M phosphate buffer (pH 7.8), 200 μL / well, coated at 37° C. for 4 hours, and washed.

[0096] (2) Detect cuvette closure

[0097] 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.

[0098] (3) Detection of cuvette drying

[0099] 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.

[0100] 2. Preparation of fluorescently labeled antibodies

[0101] (1) Pretreatment of fluorescent particles

[0102] Take 1mg of carboxyl time-resolved fluorescent microspheres (200nm, 0.1ml, 10mg / mL, Bangslab company), add 60ul 500mM MES (2-(N-morpholine)ethanesulfonic acid) buffer, pH 6.0, add 0.2mg 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add 0.4mg N-hydroxysuccinimide (...

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Abstract

The invention discloses a cup-type time-resolved fluorescent analysis method and kit for NT-proBNP based on microspheres. The method comprises the following steps: coating the interior surface of a detection reaction cup with a NT-proBNP monoclonal or polyclonal antibody; subjecting time-resolved fluorescent microspheres and the NT-proBNP monoclonal or polyclonal antibody to crosslinking via a covalent bond so as to prepare a fluorescence-labeled antibody; adding a to-be-detected NT-proBNP sample into the coated detection reaction cup, then adding the fluorescence-labeled antibody and carrying out incubation at 25 to 40 DEG C; carrying out cleaning to remove unbounded antigen and fluorescence-labeled antibody and testing a fluorescence signal in the detection reaction cup under the condition of excitation by exciting light with a wavelength of 340 to 380 nm; and comparing the fluorescence signal with a standard curve so as to obtain the concentration of NT-proBNP in the to-be-detected NT-proBNP sample. The method and kit provided by the invention can complete detection in short detection time, shortens detection time and improves detection sensitivity.

Description

technical field [0001] The invention relates to the technical field of NT-proBNP detection, in particular to a microsphere-based cup-type time-resolved fluorescent NT-proBNP 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, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/533G01N33/543G01N21/64
CPCG01N33/577G01N21/6408G01N33/533G01N33/54313
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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