Immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and preparation method of immunochromatography reagent strip

A technology for the quantitative detection of hydroxyvitamins, applied in the biological field, can solve problems such as the low degree of automation of ELISA, the complicated operation of liquid chromatography mass spectrometry, and the environmental pollution of radioimmunoassay, and achieve the universal detection and sensitivity of human vitamin D. High and reduce the effect of clinical blind drug use

Inactive Publication Date: 2015-07-01
PRO MED BEIJING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The degree of automation of enzyme-linked immunosorbent assay is not high, and it is greatly affected by human factors
Radioimmunoassay has the problem of environmental pollution; liquid chromatography mass spectrometry is complicated to operate and takes a long time; while chemiluminescence method has high sensitivity, but the detection cost is high, and a specific chemiluminescence instrument is required. These reasons lead to its limited application range

Method used

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  • Immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and preparation method of immunochromatography reagent strip
  • Immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and preparation method of immunochromatography reagent strip
  • Immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and preparation method of immunochromatography reagent strip

Examples

Experimental program
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Effect test

Embodiment 1

[0046] A test strip for quantitative detection of 25-hydroxyvitamin D, comprising a sample loading area 1, a conjugate release area 2, a reaction area 3 and a water absorption area 4 arranged in sequence; the conjugate release area is coated with mouse anti-human 25-hydroxyl A glass cellulose membrane of vitamin D monoclonal antibody, the mouse anti-human 25-hydroxyvitamin D monoclonal antibody is coated by colloidal gold, quantum dots or up-conversion luminescent material UCP; the reaction area is divided into a test area T and a control area C, The test area is coated with 25-hydroxyvitamin D-coupled protein, and the control area is a nitrocellulose membrane coated with goat anti-mouse IgG antibody.

[0047] Further, the quantitative detection test strip also includes a PVC backing plate (5), and the sample loading area, conjugate release area, reaction area, and water absorption area are sequentially overlapped and pasted on the PVC backing plate.

[0048] Further, the prep...

Embodiment 2

[0059] Example 2 : Preparation of the conjugate-releasing region

[0060] Add 2μmol colloidal gold (or quantum dots or up-conversion luminescent material UCP) and 1mg mouse anti-human 25-hydroxyvitamin D monoclonal antibody to 1mL pH7.4, 50mM phosphate buffer, shake gently at room temperature for 2 hours, then Centrifuge at 12000r / min for 30min, and take the precipitate.

[0061] Colloidal gold (or quantum dots or up-conversion luminescent material UCP) labeled mouse anti-human 25-hydroxyvitamin D monoclonal antibody, spread 20cm according to 1ml solution 2 Spray on the glass cellulose film with a film sprayer, place it at a temperature of 20-30°C, and dry it at a relative humidity of 30-35% for 5 hours to obtain a conjugate release area.

Embodiment 3

[0062] Example 3: Preparation of the reaction zone

[0063] Use 0.01M pH 7.4 phosphate buffer to prepare 25-hydroxyvitamin D-coupling protein into a solution of 0.8~1.6mg / ml, and coat it on a nitrocellulose membrane with a spray film instrument at a volume of 1~1.5μl / cm The area to be tested; another goat anti-mouse IgG antibody was formulated into a solution of 1-3 mg / ml, and the control area was coated with a film sprayer at an amount of 1-1.5 μl / cm; Dry at 38°C for 8-12 hours to prepare a nitrocellulose membrane coated with a detection area of ​​25-hydroxyvitamin D-coupled protein and a control area coated with goat anti-mouse IgG.

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Abstract

The invention relates to the technical field of biology, and discloses an immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and a preparation method of the immunochromatography reagent strip. The immunochromatography reagent strip for quantitative detection of the 25-hydroxyvitamin D comprises a sample adding region, a conjugate release region, a reaction region and a water absorption region, which are sequentially arranged; the conjugate release region is a glass cellulose membrane enveloped with colloidal gold or a quantum dot or an up-conversion luminescent material UCP-marked mouse-anti-human 25-hydroxyvitamin D monoclonal antibody; the reaction region is a test region and a control region; the test region is enveloped with 25-hydroxyvitamin D-coupling protein; and the control region is enveloped with a nitrocellulose membrane of a goat anti-mouse IgG antibody. The immunochromatography reagent strip is capable of accurately, quantitatively, rapidly and sensitively detecting the 25-hydroxyvitamin D in human whole blood, serum and plasma; the required instrument is different from a traditional large expensive instrument for a clinical laboratory; and only one corresponding miniature immunity analyzer is required, therefore, real-time detection is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an immunochromatographic reagent strip for quantitatively detecting 25-hydroxyvitamin D and a preparation method thereof. Background technique [0002] Vitamin D is a steroid derivative and is a fat-soluble vitamin. Only a small part of vitamin D in the human body comes from food, and 95% of the vitamin D needed in the body comes from the skin produced by ultraviolet rays in the sun. Vitamin D enters the liver through blood circulation, is converted into 25-hydroxyvitamin D by 25-hydroxylase, and then converted into 1,25-dihydroxyvitamin D by the α-hydroxylase system in the mitochondria of renal proximal tubular epithelial cells Vitamin D, which is the most biologically active form of vitamin D, promotes the synthesis of intestinal calcium-binding proteins. However, 25-hydroxyvitamin D is the main circulating form in metabolism, which can reflect the body's vitamin D storage level ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82G01N33/577G01N33/558
CPCG01N33/82G01N33/558
Inventor 李西兰平龙飞郝存辛玉龙陈芳芳余秀华高巍巍潘志红马康乐
Owner PRO MED BEIJING TECH
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