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25 hydroxyl vitamin d detection kit and preparation method thereof

A technology for detecting kits and hydroxyvitamins, which is applied in the field of clinical in vitro diagnosis and medical immunology, and can solve the problems of large influence of human factors, complicated operation of liquid chromatography mass spectrometry, environmental pollution of radioimmunoassay, etc.

Active Publication Date: 2013-06-19
BEIJING STRONG BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • 25 hydroxyl vitamin d detection kit and preparation method thereof
  • 25 hydroxyl vitamin d detection kit and preparation method thereof
  • 25 hydroxyl vitamin d detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Preparation of 25-hydroxyvitamin D detection kit

[0030] 1. The preparation process of the first reagent is as follows:

[0031] The first reagent composition:

[0032]

[0033] With 0.15M NaCl, 0.1%NaN 3 , 0.1% BSA in 50mM MES solution, dilute vitamin D-binding protein antibody and 25-hydroxyvitamin D antibody at room temperature to a final antibody concentration of 0.2%.

[0034] 2. The preparation process of the second reagent is as follows:

[0035] a) Dilute 4ml of human 25-hydroxyvitamin D antigen (10mg / ml) with anhydrous acetone at room temperature to a final antigen concentration of 2mg / ml;

[0036] b) Weigh 20mg of carbonyldiimidazole into the above solution, mix well, and react in a shaker at 30°C and 150rpm for 0.5-2 hours, for example, 1 hour;

[0037]c) Dilute 1ml of 223nm polystyrene latex particles with 20mM MES solution (pH5.0) at room temperature to a latex concentration of 4%;

[0038] d) Weigh 10 mg of diaminodipropylimine and d...

Embodiment 2

[0050] Embodiment 2: comparison preparation method

[0051] 1. The preparation of the first reagent is the same as in Example 1.

[0052] 2. The preparation process of the second reagent is as follows:

[0053] a) Dilute 4ml of human 25-hydroxyvitamin D antigen (10mg / ml) with 20mM MES solution (pH5.0) at room temperature to a final antigen concentration of 2mg / ml;

[0054] b) Dilute 1ml of 223nm polystyrene latex particles with 20mM MES solution (pH5.0) at room temperature to a latex concentration of 4%;

[0055] c) Weigh 1.5 mg of diaminodipropylimine into b) and stir for 10 to 60 minutes, for example, 30 minutes;

[0056] d) Add the polystyrene latex particles obtained in c) dropwise into a), mix well, and react in a 150rpm shaker at 30°C for 1-4 hours, for example, 3 hours;

[0057] e) Centrifuge the liquid obtained in d), wash it 2-3 times with deionized water, remove the supernatant, and obtain latex particles;

[0058] f) Dilute the latex particles obtained in e) to ...

Embodiment 3

[0061] Embodiment 3: Drawing of 25 hydroxyvitamin D calibration curve

[0062] The assay steps of the kit of the present invention are as follows:

[0063]

[0064] Taking the concentration of calibrator as the horizontal axis, the corresponding △OD 570 As the vertical axis, use nonlinear fitting, such as spline to draw a standard curve, such as figure 1 .

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Abstract

The invention relates to a 25 hydroxyl vitamin D detection kit and a preparation method thereof. A reagent comprises a first reagent and a second reagent and selectively further comprises a correction product. The first reagent comprises a 25 hydroxyl vitamin D antibody, a vitamin D binding protein antibody and buffer liquid. The second reagent comprises a micro-balloon combined with a 25 hydroxyl vitamin D antigen and the buffer liquid. The 25 hydroxyl vitamin D and the antigen on a surface of the micro-balloon are combined with the 25 hydroxyl vitamin D antibody in a competing mode so that turbidity generated by reaction of the micro-balloon combined with the 25 hydroxyl vitamin D and the 25 hydroxyl vitamin D antibody is reduced. Content of the 25 hydroxyl vitamin D in a sample can be calculated according to reducing degree of the turbidity.

Description

technical field [0001] The invention relates to the fields of clinical in vitro diagnosis and medical immunology; in particular, it relates to an immunological detection reagent; further, the invention relates to a 25-hydroxyvitamin D detection kit and a preparation method thereof. Background technique [0002] Vitamin D, also known as anti-rickets vitamin, can be divided into vitamin D2 and vitamin D3. Vitamin D2 is mostly contained in plant foods. It is synthesized by plant ergosterol through sunlight. Vitamin D3 can be synthesized by human skin and fat tissue from 7-dehydrocholesterol through sunlight. Vitamin D2 is a fat-soluble vitamin. Vitamin D from food is absorbed through the small intestine together with fat, forms chylomicrons with the assistance of bile, enters the blood from lymphatic vessels, and is transported to the liver together with self-synthesized vitamin D3. In the liver, 25-hydroxyvitamin D is formed through the action of the monooxygenase system (25-...

Claims

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Application Information

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IPC IPC(8): G01N33/82G01N33/531
CPCG01N33/82C07K16/26
Inventor 刘鹤张小锐蔡华雅刘希
Owner BEIJING STRONG BIOTECH INC
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