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Kit for quantitatively detecting myeloperoxidase, and preparation method thereof

A technology for quantitative detection of myeloperoxidase, which is applied in the field of kits and preparations for quantitative detection of myeloperoxidase, can solve the problems of poor specificity and low sensitivity, achieve high specificity, high sensitivity, and ensure sensitivity Effect

Inactive Publication Date: 2018-08-24
浙江艾明德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The invention provides a kit and preparation method for quantitative detection of myeloperoxidase, which overcomes the shortcomings of low sensitivity and poor specificity in the prior art

Method used

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  • Kit for quantitatively detecting myeloperoxidase, and preparation method thereof
  • Kit for quantitatively detecting myeloperoxidase, and preparation method thereof
  • Kit for quantitatively detecting myeloperoxidase, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of MPO Acridine Lipid-labeled Monoclonal Antibody

[0053] 1) Weigh an appropriate amount of MPO monoclonal antibody and use 0.05mol / L pH 9.5 carbonate buffer (CB) to adjust to 1mg / mL;

[0054] 2) Acridine lipid and antibody are calculated at 1:15 molar ratio, dissolved in DMF, mixed, and reacted at room temperature for 30 minutes;

[0055] 3) Transfer the reaction solution to a dialysis bag (molecular weight cut-off 8000-12000), and dialyze with 0.01M PBS with pH 7.2 for 24 hours;

[0056] 4) Add an equal volume of glycerin and 0.1% PC-300.

[0057] The acridinium ester marker may be an acridone compound, an acridine sulfonamide compound or acridinyl aminoacetic acid.

Embodiment 2

[0058] Example 2 Preparation of the MPO quantitative assay kit of the present invention

[0059] 1. Preparation of acridine lipid antibody

[0060] 1. Acridine-labeled monoclonal antibody diluent Buffer A:

[0061] Buffer A: 0.01-0.1M PBS pH 7.2, 0.1%-1%BSA, 0.2% PC-300

[0062] 2. Select the concentration of acridine-labeled monoclonal antibody:

[0063] The working concentration of acridinium-labeled monoclonal antibody is greater than 0.45 μg / mL.

[0064] 2. Preparation of MPO calibrator

[0065] Dilute the MPO antigen with 0.01M PBS pH 7.2, 0.5% BSA diluent, and prepare five kinds of calibrator with standard concentration of 0, 5, 25, 100, 500, 2000ng / mL.

[0066] 3. Preparation of antibodies labeled with acridine derivatives:

[0067] 1) Weigh an appropriate amount of MPO monoclonal antibody, and use 0.05mol / L pH 9.5 CB to adjust to 1mg / mL;

[0068] 2) Acridine derivatives and antibodies are calculated at 1:15 molar ratio, dissolved in DMF, mixed, and reacted at roo...

Embodiment 3

[0094] Example 3 Buffer Buffer

[0095] As an important component of the present invention, the buffer is a buffer for diluting biotinylated antibodies, antibodies labeled with acridine derivatives, and streptavidin-coated magnetic particles, using a combined formula of BSA, trehalose and casein, Improve the non-specific physical adsorption of immunoglobulin by the magnetic particles, further reduce the specific binding, and improve the analytical sensitivity of the reagent.

[0096] Buffer A: 0.01-0.1M PBS pH 7.2, mass ratio 0.1%-1%BSA, volume ratio 0.2% PC-300;

[0097] Buffer B: 0.01-0.1M PBS pH 7.2, mass ratio 0.1%-1%BSA, mass ratio 0.1%-5% trehalose, mass ratio 0.5%-1% casein, volume ratio 0.2% PC-300.

[0098] Analytical Sensitivity=2*(0 value RLU SD)*5 / (Calibrator RLU mean - 0 value RLU mean)

[0099]

[0100] Use Buffer B as the diluent of acridinium ester-MPO antibody conjugate (AE-MPO) 2#, AE-MPO4# (2#, 4# are antibody numbers), magnetic particle-streptavidin (M...

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Abstract

The invention provides a kit for quantitatively detecting myeloperoxidase. The kit comprises a myeloperoxidase calibrator, a magnetic particle coated with streptavidin, an acridine derivative-labeledmyeloperoxidase monoclonal antibody, a biotin-labeled myeloperoxidase monoclonal antibody, a chemiluminescent substrate, a quality control substance and a cleaning solution. A preparation method of the kit comprises the following steps: preparing the calibrator from a pure peroxidase raw material; preparing the acridine derivative-labeled antibody; preparing a biotinylated antibody; coating a magnetic particle with streptavidin; sub-packaging the calibrator, a marker mixture solution and the chemiluminescent substrate; and performing assembling to obtain the finished product. The kit has the advantages of high sensitivity, good specificity, high accuracy of a quantitative detection result, low use cost, and easiness in promotion and application.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a kit for quantitatively detecting myeloperoxidase and a preparation method. Background technique [0002] Myeloperoxidase (MPO) is the main component of azurophilic blue granules in leukocytes and is secreted when leukocytes are activated in innate immunity. MPO is a member of the hemoglobin peroxidase superfamily, a glycosylated tetrameric heme protein with a relative molecular weight of 140KDAa, containing two identical subunits, and each subunit is composed of a heavy chain and a light chain through two Formed by sulfur bonds, MPO exists in three subtypes in myeloid cells, namely MPOⅠ, MPOⅡ, and MPOⅢ. The differences in the structure of the three subtypes are mainly in the heavy chain, which ultimately leads to their relative molecular mass, hydrophobicity, etc. The differences in function of the three subtypes are still inconclusive. [0003] During the occurrence and developmen...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/573G01N21/76
CPCG01N21/76G01N33/573G01N33/577G01N2800/32
Inventor 陈志强胡国富朱炎
Owner 浙江艾明德生物科技有限公司
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