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

A time-resolved fluorescence and analysis method technology, applied in the field of cup-type time-resolved fluorescence FABP analysis methods and kits based on microspheres, can solve the problems of unsatisfactory precision, complicated detection steps, poor precision, etc. The effect of reducing the resistance effect, shortening the detection time, and improving the 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

[0010] The purpose of the present invention is to overcome the shortcomings of the above-mentioned existing fluorescent immunochromatography method for measuring FABP detection precision and the time-resolved fluorescence detection method needs a dissociation enhancement process, complicated detection steps, long time, poor precision, etc., to provide a micro-based Ball cup time-resolved fluorescence FABP analysis method and kit

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1. Preparation of detection cuvette

[0058] (1) Detection cuvette coating

[0059] The h-FABP antibody 2302 (Medix 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 the plate was washed.

[0060] (2) Detect cuvette closure

[0061] 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.

[0062] (3) Detection of cuvette drying

[0063] 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.

[0064] 2. Preparation of fluorescently labeled antibodies

[0065] (1) Pretreatment of fluorescent particles

[0066] Take 1mg of carboxyl time-resolved fluorescent microspheres (300nm, 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-ethylcarbod...

Embodiment 2

[0093] 1. Preparation of detection cuvette

[0094] (1) Detection cuvette coating

[0095] The h-FABP antibody 2302 (Medix 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 the plate was 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 (300nm, 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-ethylcarbod...

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Abstract

The invention discloses a cup type time-resolved fluorescence FABP 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 h-FABP monoclonal antibodies or polyclonal antibodies; the time-resolved fluorescence microspheres and the FABP monoclonal antibodies or polyclonal antibodies are subjected to covalent bond cross-linking to prepare fluorescence-labeled antibodies; an h-FABP 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 h-FABP concentration of the h-FABP 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 FABP detection, in particular to a microsphere-based cup-type time-resolved fluorescence FABP analysis method and a kit. Background technique [0002] Cardiovascular disease is a serious disease that endangers human health and life, and has become the number one killer of human health in the 21st century. In August 2009, Bayer Pharmaceuticals reported at the annual meeting of the European Society of Cardiology that about 17.5 million people died of cardiovascular diseases in the world in 2005, and this number is expected to climb to 20 million in 2015, and about half of the cases occurred in the Asia-Pacific region. area. The improvement of living conditions and the change of lifestyle have made the risk of cardiovascular disease in Chinese people surpass that of developed countries such as the United States. [0003] In 1990, the mortality rate of cardiovascular diseases among urban residents in my cou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/324
Inventor 石晓强徐建新李福刚周奕璇杨晶
Owner SHANGHAI UPPER BIO TECH PHARMA
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