25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof

A technology of hydroxyvitamins and kits, applied in the direction of biological testing, material inspection products, etc., to achieve the effects of quantitative accuracy, high sensitivity, and simple operation

Active Publication Date: 2013-03-27
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there is still a lack of a kit for detecting the content of 25-hydroxyvitamin D3 in the human body that is simple, fast, quantitatively accurate, highly sensitive or specific

Method used

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  • 25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof
  • 25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof
  • 25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Preparation of Preliminary Example 1 25-Hydroxyvitamin D3-BSA Conjugate

[0047] ① Weigh 2.0 mg of 25-hydroxyvitamin D3-hemisuccinate (purchased from Shanghai Hanzun Biological Company), 0.8 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 2.0 mg of N-hydroxysuccinimide (NHS) were added to a round-bottomed flask filled with 1 mL of anhydrous chloroform, and stirred at room temperature for 2 h under nitrogen protection;

[0048] ② After the reaction is terminated, add 1mL of 3mg / mL BSA solution to the round bottom flask, and stir and react overnight at room temperature under the protection of nitrogen;

[0049] ③The above reaction solution was dialyzed with 0.01mol / L PBS with pH 7.2 at 4°C for 48 hours, the medium was changed 6 times in the middle, and the solution was changed once every 8 hours. TM Desalting column desalting and purification twice, vacuum freeze-drying to obtain 25-hydroxyvitamin D3-BSA conjugate 25-hydroxyvitamin D3-hemisuccinate-BSA.

Embodiment 2

[0050] Preliminary Example 2 Preparation of 25-hydroxyvitamin D3 polyclonal antibody

[0051] ①Use 25-hydroxyvitamin D3-hemisuccinate-BSA to immunize 3-month-old male Japanese white rabbits weighing 1.5kg. For the first immunization, dissolve 25-hydroxyvitamin D3-hemisuccinate- BSA was mixed with an equal amount of Freund's complete adjuvant (FCA) to fully emulsify, the dose of immune agent was 1mg (0.5mL) per mouse, and injected subcutaneously in 4-6 parts on the back;

[0052] ② Two weeks after the first immunization, 25-hydroxyvitamin D3-hemisuccinate-BSA was also dissolved in sterile PBS solution and mixed with the same amount of incomplete adjuvant (FIA) to fully emulsify the rabbits, and the immunization dose was 500 μg ( 0.5mL) per mouse, the injection site is the same as before.

[0053] ③ All the rabbits were immunized 6 times with an interval of 2 weeks. After the 4th immunization, the titer and specificity of rabbit antiserum were determined by indirect ELSIA and i...

Embodiment 3

[0054] Preliminary Example 3 Preparation of Polyclonal Antibody Latex Particles Coated with 25-Hydroxyvitamin D3

[0055] ①Weigh 1.0g of dry polystyrene latex particles (purchased from PolyMicrospheres), add 100mL of 0.12M phosphate buffer (pH 7.5) to disperse, then add 20mL of 0.01M 6-aminocaproic acid solution and 40mg of 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), stirred at room temperature for 3 hours, centrifuged to discard the supernatant, and washed the precipitate twice with 0.12M phosphate buffer to prepare a graft Carboxylated polystyrene latex with aminocaproic acid arms, then dilute and disperse the precipitated latex with 100mL 0.12M phosphate buffer to obtain carboxylated modified polystyrene latex solution;

[0056] ② Dissolve 250 μg of 25-hydroxyvitamin D3 polyclonal antibody freeze-dried product in 0.5 mL deionized water to obtain antibody solution;

[0057] ③ Dilute the antibody solution prepared in step ② with 25mL 0.12M phosphate buffer, add 25m...

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Abstract

The invention discloses a 25-hydroxy-vitamin D3 detection kit, as well as a preparation method and applications thereof. The detection kit consists of a reagent I and a reagent II which are independent from each other, wherein the reagent I comprises the following components: a 25-hydroxy-vitamin D3-BSA conjugate, a biobuffer, a chelating agent, a surface active agent, a coagulant, a preservative, a stabilizer and water; the reagent II comprises the following components: rubber latex particles coated by a 25-hydroxy-vitamin D3 antibody, a biobuffer, a chelating agent, a surface active agent, a suspending agent, a preservative, a sealing agent, a stabilizer and water. The detection kit disclosed by the invention is high in detection sensitivity, high in specificity, accurate in quantification and wide in linear range, and is especially suitable for the detection of samples with extremely low 25-hydroxy-vitamin D3 content. In addition, the detection kit disclosed by the invention does not need to dilute the samples in advance in the detection process, facilitates the clinical use, and is easy to operate, low in detection cost and suitable for various full-automatic biochemical analyzers.

Description

technical field [0001] The invention relates to a kit for detecting the content of 25-hydroxyvitamin D3 in the human body and a preparation method thereof. The invention also relates to a method for using the 25-hydroxyvitamin D3 assay kit in detecting the content of 25-hydroxyvitamin D3 in the human body, which belongs to The field of detection of 25-hydroxyvitamin D3 content. Background technique [0002] 25-Hydroxyvitamin D3 is one of the main forms of vitamin D stored in the human body, and it is one of the indispensable substances for normal metabolic activities of the human body. Overall, two-thirds of the world's population is deficient in vitamin D, with vitamin D deficiency more likely to occur in children, the elderly, and pregnant women. The lack of vitamin D in the body greatly increases the possibility of children suffering from rickets and the elderly suffering from osteoporosis. A large number of studies in recent years have shown that vitamin D deficiency d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82
Inventor 华权高沈鹤霄许可闫彩霞常向博舒芹余箭翔
Owner CUSABIO TECH LLC
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