Detection reagent for detecting 25-hydroxy vitamin D, as well as preparation method and application of detection reagent

A technology of hydroxyvitamin and detection agent, applied in the field of biological immunology detection, can solve the problems of decreased activity, instability, harsh storage conditions, etc., and achieve the effect of enhancing stability and reducing degrees of freedom

Active Publication Date: 2014-10-01
SHENZHEN NEW INDS BIOMEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the detection process, the stability of the 25-hydroxyvitamin D antigen derivative labeled ABEI is the most important factor affecting the stability of the detection, and the 25-hydroxyvitamin D antigen derivative is a kind of poor stability. etc. will lead to a decrease in activity, so the reagents in the above detection kits have poor stability and are prone to decrease in activity
[0006] Therefore, the instability of the 25-hydroxyvitamin D antigen derivatives used in the immunological competition method results in the inability of long-term stable storage of the 25-hydroxyvitamin D immunological method assay reagents, thereby affecting its use and detection results, and storage Harsh conditions and inconvenient storage and transportation have become a bottleneck for the large-scale commercial application of such methods

Method used

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  • Detection reagent for detecting 25-hydroxy vitamin D, as well as preparation method and application of detection reagent
  • Detection reagent for detecting 25-hydroxy vitamin D, as well as preparation method and application of detection reagent
  • Detection reagent for detecting 25-hydroxy vitamin D, as well as preparation method and application of detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of 25-hydroxyvitamin D detection agent M

[0037] 1.1) Formation of conjugates of 25-hydroxyvitamin D antigen derivatives and protein carriers

[0038] Dissolve 1 mg of 25-hydroxyvitamin D antigen derivative M' (m=0, n=4 in formula (I), 0.262 mg of succinic anhydride and 0.37 μL of triethylamine (catalyst) in anhydrous DMF, Under the protection of gas, react in the dark at room temperature for 4 hours; then add 0.25 mg NHS and 1.3 mg of DCC, and under the protection of argon, let stand overnight in the dark at room temperature; then slowly add the solution dropwise to bovine gamma globulin (20 mg bovine gamma Dissolve protein in 2ml0.1mol / L NaHCO 3 ) solution, stirred at room temperature for 4 h; centrifuged to remove the precipitate, and the supernatant was purified with a G25 gel column, and the collected protein peak (eluent: 0.1mol / L phosphate buffer saline (PBS), pH7.4), namely A conjugate of 25-hydroxyvitamin D antigen derivative M' and pro...

Embodiment 2

[0043] Example 2 Detection of 25-hydroxyvitamin D

[0044] The 25-hydroxyvitamin D detection agent prepared in Example 1 was used to detect the content of 25-hydroxyvitamin D in the sample by means of chemiluminescence detection.

[0045] 2.1) Labeling of 25-hydroxyvitamin D antibody

[0046] Take 1mg of 25-hydroxyvitamin D antibody, adjust its volume to 1ml with 0.1mol / L carbonate buffer (pH9.5), put it into a dialysis bag, and dialyze in pH9.5 carbonate buffer 1 hour. Take out the dialyzed 25-hydroxyvitamin D antibody, add 100 μg ABEI-succinamic acid-NHS, and shake at room temperature for 1.5 hours.

[0047] Install the G-25 gel column, wash it with purified water, and then use PBS buffer with a pH value of 7.4 for equilibrium elution. After the equilibration and elution of the G-25 gel column, the 25-hydroxyvitamin D antibody labeled with ABEI was purified through the column, and then the peak protein solution was collected. The collected protein solution was added to a...

Embodiment 3

[0064] Example 3 Accelerated stability test of 25-hydroxyvitamin D immunological detection kit

[0065] The 25-hydroxyvitamin D immunological detection kit includes the 25-hydroxyvitamin D detection agent prepared in Example 1, the ABEI-labeled 25-hydroxyvitamin D monoclonal antibody, the replacement agent, and the 25-hydroxyvitamin D high-point calibrator and low point calibrator.

[0066] Select three different concentrations of 25-hydroxyvitamin D sample solutions (1, 2, 3), and use the above-mentioned kits to measure them with the Maglumi series chemiluminescence analyzer of New Industry Biomedical Engineering Co., Ltd. at 37°C. The luminous intensities of these three sample solutions were measured once a day for 30 consecutive days; wherein, during the entire test process, the three 25-hydroxyvitamin D sample solutions used for testing were preserved in a frozen storage environment, and the used The kit has been stored at 37°C. see results Figure 4 (1A, 2A, 3A).

[0...

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Abstract

The invention provides a detection reagent for detecting hydroxy vitamin D, a preparation method of the detection reagent and application of the detection reagent to immunological detection of 25-hydroxy vitamin D. The detection reagent comprises a conjugate substance and a magnetic sphere, wherein the conjugate substance is formed by 25-hydroxy vitamin D antigen derivatives and protein carriers; the magnetic sphere is coated with the conjugate substance. By modifying the 25-hydroxy vitamin D antigen derivatives, the stability of the produced reagent is obviously improved, and the specificity and accuracy in the detection of the 25-hydroxy vitamin D are also improved. The invention further provides a kit for detecting 25-hydroxy vitamin D; the kit comprises the detection reagent, and the kit is good in stability, high in accuracy and high in universality.

Description

technical field [0001] The invention relates to the field of biological immunology detection, in particular to a detection agent for detection of 25-hydroxyvitamin D and its preparation method and application. Background technique [0002] Vitamin D is a fat-soluble vitamin. Only a small portion of vitamin D in the human body comes from food (<10%), and 90% of the vitamin D required in the body comes from the skin produced by ultraviolet rays in the sun. The main forms of vitamin D that occur in the body are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The nutritional sources of vitamin D2 are mainly some vegetables, yeast and fungal foods, and vitamin D3 is transformed from subcutaneous 7-dehydrocholesterol under the action of sunlight. [0003] Food sources and self-synthesized vitamin D2 and vitamin D3 enter the liver through blood circulation, and are converted into 25-OH vitamin D2 and 25-OH vitamin D3 respectively under the action of 25-hydroxylas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82G01N33/532
CPCG01N33/54326G01N33/82
Inventor 饶微李婷华杜凯袁锦云罗凯余慧玲
Owner SHENZHEN NEW INDS BIOMEDICAL ENG
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