Chemiluminescence detection kit for Es (E-selectin) and preparation method of kit

A chemiluminescence detection and chemiluminescence technology, which is applied in chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, can solve problems such as complex reaction kinetics, unstable results, and many influencing factors. Achieve the effects of no reduction in photon yield, good reagent stability, and low background luminescence

Inactive Publication Date: 2018-05-29
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

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preparation example Construction

[0025] The invention provides a chemiluminescent detection kit for E-selectin and a preparation method thereof, a magnetic particle-coupled E-selectin (Es) capture antibody, and an acridinium ester-labeled E-selectin (Es) detection antibody. The kit also includes E-selectin 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 acridinium ester-labeled E-selectin ...

Embodiment 1

[0034] Example 1: The composition and preparation method of a chemiluminescent detection kit for E-selectin

[0035] 1. Assembly of the kit

[0036] Magnetic particle-coupled E-selectin monoclonal antibody;

[0037] E-selectin (Es) monoclonal antibody labeled with acridinium ester;

[0038] E-selectin series 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 E-selectin 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 μg E-selectin monoclonal antibody so t...

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 of chemiluminescence pre-excitation solution A, add 100 μL of chemiluminescence excitation solution B after 1 second, and measure its relative luminous intensity. The content of E-selectin in the sample is proportional to its relative luminous intensity.

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Abstract

The inventon discloses a chemiluminescence detection kit for Es (E-selectin) and a preparation method of the kit. The kit comprises a magnetic particle coupled Es capturing antibody, an acridinium ester labeled Es detection antibody, an Es calibrator, a chemiluminescence pre-excitation liquid A, a chemiluminescence pre-excitation liquid B and a cleaning liquid. The kit combines the chemiluminescence technology with immune magnetic particles and provides a nearly homogeneous reaction system. Compared with the prior art, the established direct chemiluminescence method has high sensitivity and high specificity and is accurate and rapid, the detection time is short, the detection result has higher accuracy and repeatability, and the kit is applicable to various luminescence detecting instruments.

Description

technical field [0001] The invention belongs to the field of immunological detection and analysis, in particular to an E-selectin immunochemiluminescent detection kit and a preparation method thereof. Background technique [0002] E-selectin (Es), known as "endothelial cell leukocyte adhesion molecule" (EL-AM-1), has now been uniformly named Es or CD 62 e. Like other members of the selectin family, Es is a single-chain glycoprotein with a relative molecular mass of 115x10 3 , which consists of a series of protein domain fragments, including an amino-terminal C-type lectin (leotin) factor-like fragment, an epidermal growth factor (EGF)-like fragment, 6 shorter common repeats (SCR) (or complement regulation / Binding protein repeat sequences, each sequence contains about 60 amino acids), a single transmembrane domain and a cytoplasmic domain. Compared with other selectins, the main structural features of Es are: multiple repeats of the complement regulatory type combine with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N21/76
CPCG01N33/6893G01N21/76G01N33/54326G01N2800/323
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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