Neutral granulocyte elastase chemiluminescence detection kit and preparation method thereof

A technology of chemiluminescence detection and elastase, which is applied in chemiluminescence/bioluminescence, biological testing, and analysis by making materials undergo chemical reactions, can solve problems such as complex reaction kinetics, unstable results, and difficult substrates , to achieve the effect of low cost, good reagent stability and rapid response

Inactive Publication Date: 2018-05-15
太原瑞盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme used in ELISA is horseradish peroxidase, and the main disadvantage of using horseradish peroxidase is that luminol will also be absorbed by H in the absence of horseradish peroxidase. 2 o 2 Oxidation is self-luminescent, the background is relatively high, which affects the signal-to-noise ratio, the reaction kinetics is complex, there are many influencing factors, the result is not stable enough, and it is not easy to obtain a substrate with high sensitivity and long plateau period

Method used

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Examples

Experimental program
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Effect test

preparation example Construction

[0025] The invention provides a chemiluminescent detection kit and preparation method for neutrophil elastase, including magnetic particle-coupled neutrophil elastase (NE) capture antibody, acridinium ester-labeled neutrophil elastase ( NE) detection antibody. The kit also includes neutrophil elastase calibrator, chemiluminescence pre-excitation solution A, chemiluminescence excitation solution B and cleaning solution.

[0026] Specifically, in the process of preparing the magnetic particle-coupled capture antibody in the present invention, the antibody-coupled buffer is a buffer with a pH value of 5.0-6.0 and a concentration of 20-200 mmol / L MES.

[0027] Specifically, in the process of preparing the magnetic particle-coupled capture antibody in the present invention, the blocking buffer is a buffer containing 1% BSA.

[0028] Specifically, in the process of preparing the acridinium ester-labeled detection antibody in the present invention, the buffer solution of the acridin...

Embodiment 1

[0034] Example 1: Construction and preparation method of a chemiluminescence detection kit for neutrophil elastase

[0035] 1. Assembly of the kit

[0036] Magnetic particle-conjugated monoclonal antibody to neutrophil elastase;

[0037] Acridinium ester-labeled neutrophil elastase (NE) monoclonal antibody;

[0038] Neutrophil elastase calibrator solution;

[0039] Chemiluminescence pre-excitation solution A and chemiluminescence excitation solution B;

[0040] Cleaning fluid.

[0041] 2. Preparation of Magnetic Microparticle Suspension Conjugated to Neutrophil Elastase Monoclonal Antibody

[0042] (1) Take 1 mg of carboxyl magnetic particles in a 0.5 mL centrifuge tube, add 200 μL of MES buffer solution with a concentration of 0.1 mol / L, vortex and mix, place on a magnetic stand, and let it stand for 5 minutes to make the magnetic particles and liquid Separate, discard the supernatant, wash 3 times, then add 200 μL of MES (pH 6.0) buffer, and vortex.

[0043] (2) Add 15 μ...

Embodiment 2

[0062] Embodiment 2: detection and result analysis with kit

[0063] (1) Add 50 μL of the sample to be tested into the cuvette, then add 150 μL of magnetic particle coupling suspension, shake and mix, and incubate at 37°C for 8 min.

[0064] (2) Separate and wash 3 times. Shake the washed reaction vessel sufficiently to disperse the magnetic particles. .

[0065] (3) Add 150 μL of acridinium ester marker into the cuvette, shake to mix, and incubate at 37°C for 7 min.

[0066] (4) Separate and wash 3 times. Shake the washed reaction vessel sufficiently to disperse the magnetic particles.

[0067] (5) Add 100 μL chemiluminescence pre-excitation solution A, add 100 μL chemiluminescence excitation solution B after 1 second, and measure the relative luminous intensity. The content of neutrophil elastase in the sample is proportional to its relative luminous intensity.

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Abstract

The invention discloses a neutral granulocyte elastase chemiluminescence detection kit and a preparation method thereof. The kit comprises a magnetic particle coupled neutral granulocyte elastase (NE)capture antibody, acridinium ester-labelled neutral granulocyte elastase (NE) detection antibody, a neutral granulocyte elastase calibration product, a chemiluminescence pre-excitation solution A, achemiluminescence pre-excitation solution B, and a cleaning solution. The kit combines a chemiluminescence technology and immunization magnetic particles, and provides a reaction system which approaches to a homogeneous phase. Compared with the prior art, the established direct chemiluminescence method has the advantages of high sensitivity, strong specificity, accuracy and rapidity, and short detection time, the detection result has higher accuracy and repetition performance, and the kit can be used for various luminescence detection apparatuses.

Description

technical field [0001] The invention belongs to the field of immunological detection and analysis, in particular to an immunochemiluminescent detection kit for neutrophil elastase and a preparation method thereof. Background technique [0002] Neutrophil elastase (NE) is a single-chain glycoprotein with a molecular weight of 30,000 and composed of 218 amino acids, belonging to the serine protease family. It is derived from neutrophils and is also present in small amounts in monocytes. The concentration of elastase in neutrophils exceeds 5mM, and each neutrophil contains about 400 elastase-positive granules, and the total amount of cells is estimated to be above 3pg, and is strictly controlled within the neutrophil azurophilic body within the particle. After the neutrophils are activated, a variety of proteolytic enzymes are released from the azurophilic blue granules, including serinase, elastase and other proteases, and elastase is a protein with high content, wide range ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/68G01N33/543G01N33/532G01N33/577G01N21/76
CPCG01N33/573G01N21/76G01N21/763G01N33/532G01N33/54326G01N33/577G01N33/6893G01N2333/96433G01N2446/90G01N2800/324
Inventor 曹晶刘丽青胡雪婷常燕杜爱铭徐兵
Owner 太原瑞盛生物科技有限公司
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