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Preparation method of human myeloperoxidase immunochromatographic test strip

A myeloperoxidase and immunochromatographic test paper technology, which can be used in analytical materials, measuring devices, instruments, etc., can solve problems such as insufficient accuracy and sensitivity

Inactive Publication Date: 2017-08-18
郑乐民 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, immunochromatographic test strips are used to detect the concentration of MPO in blood, but although the immunochromatographic test strips prepared by traditional methods are easy and fast to use, they have the disadvantages of insufficient accuracy and sensitivity.

Method used

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  • Preparation method of human myeloperoxidase immunochromatographic test strip
  • Preparation method of human myeloperoxidase immunochromatographic test strip
  • Preparation method of human myeloperoxidase immunochromatographic test strip

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preparation example Construction

[0025] The first aspect of the present disclosure provides a method for preparing a human myeloperoxidase immunochromatographic test strip, characterized in that the immunochromatographic test strip includes a sample pad, a labeled antibody pad, an antibody pad, and an antibody pad sequentially connected. Coated with nitrocellulose membrane and absorbent pad; the labeled antibody pad contains 60-80 μg of colloidal gold or the first monoclonal antibody of fluorescently labeled human myeloperoxidase; the preparation method includes antibody-coated nitrocellulose The preparation of plain film and the assembly of immunochromatographic test strips, the preparation of described antibody-coated nitrocellulose membrane comprises the following steps:

[0026] S1. Mix the second monoclonal antibody solution of 1-2mg / mL human myeloperoxidase and 0.3-0.7% (W / V) 3-aminopropyltriethoxysilane phosphate buffer according to volume ratio 1: (4-6) mixed evenly to obtain 3-aminopropyltriethoxysil...

Embodiment 1

[0044] Take 20mL 40μg / mL colloidal gold solution in a 50mL clean centrifuge tube, after returning to room temperature, add 0.2mol / LK 2 CO 3 Adjust the pH to 8.0, and stir at a rate of 200 rpm under dark conditions. Add 10 μg of the primary monoclonal antibody to human myeloperoxidase into 0.5 ml of borate buffer and mix well, then slowly add it dropwise into the colloidal gold, and continue stirring for 20 min. Then slowly add 10% (W / V) bovine serum albumin dropwise to a final concentration of 1% (W / V), and continue stirring for 1 h. Stand overnight at 4°C to terminate the reaction. After the reaction, centrifuge at 15,000 rpm for 1 h at 4°C, and discard the supernatant. The precipitate is suspended in the bovine serum albumin solution of 1% (W / V) of original volume 1 / 10 to obtain the solution containing the first monoclonal antibody of human myeloperoxidase labeled with colloidal gold, and the solution is sprayed with gold The film dispenser evenly sprays on the glass cel...

Embodiment 2

[0048] The europium-containing polystyrene fluorescent microspheres were diluted to a concentration of 0.04mg / ml with 0.02M pH7.4 phosphate buffer solution, and then EDC (carbodiimide) with a final concentration of 2mM and NHS with a final concentration of 5mM were added (N-hydroxysuccinimide) was reacted at room temperature for 15 minutes, and the supernatant was removed by centrifugation at 15,000 rpm. Add 1.7 μL of the first monoclonal antibody of human myeloperoxidase with a concentration of 6 mg / mL to the precipitate, react at room temperature for 4 h, and then add 2% (W / V) glycine, 1% (W / V) bovine Serum albumin was treated with 0.02M pH 7.4 phosphate buffer for 30min, centrifuged to remove the supernatant, and then 1% (W / V) bovine serum albumin 0.02M pH 7.4 phosphate was added to the precipitate The buffer solution is suspended to obtain the first monoclonal antibody solution of human myeloperoxidase labeled with europium polystyrene fluorescent microspheres. The soluti...

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Abstract

The invention discloses a preparation method of a human myeloperoxidase immunochromatographic test strip. The immunochromatographic test strip comprises a sample pad, a labeled antibody pad, an antibody-coating nitrocellulose membrane and an absorption pad which are sequentially connected; and the labeled antibody pad contains 60-80mu g of colloidal gold or fluorescence-labeled human myeloperoxidase first monoclonal antibody. The preparation method comprises the following steps: preparing the antibody-coating nitrocellulose membrane, and assembling the immunochromatographic test strip. 3-aminopropyltriethoxysilane is used during preparation of the antibody-coating nitrocellulose membrane, so that the sensitivity of the prepared human myeloperoxidase immunochromatographic test strip is improved.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular to a method for preparing a human myeloperoxidase immunochromatographic test strip. Background technique [0002] Human myeloperoxidase (myeloperoxidase, MPO) is a heme protease containing heme prosthetic group secreted by neutrophils, monocytes and macrophages of certain tissues, and is a member of the heme peroxidase superfamily one. MPO is the functional marker and activation marker of neutrophils. Activated neutrophils release a large amount of MPO, which affects the occurrence of various diseases. Studies have found that the activated neutrophils in the peripheral blood of patients with atherosclerosis increase, and MPO promotes the formation of atherosclerotic plaques and increases the instability of plaques by producing free radicals and various reactive substances. The process of atherosclerosis; MPO can be used for the differential diagnosis of early acute myeloid leukemia a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 郑乐民段学军贾志磊王洋刘明阳闫宝山
Owner 郑乐民
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