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A vitamin D synthetic antigen, its preparation method and application

A technology for synthesizing antigens and vitamins, applied in the field of immunology, can solve the problems of long detection time and low specificity, and achieve the effects of shortened detection time, rapid detection and satisfactory precision

Active Publication Date: 2016-01-06
BEIJING BOHUI INNOVATION TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this type of kit is that 25-hydroxyvitamin D3 is labeled with biotin, avidin is labeled with HRP, and the solid-phase antibody is detected by polyclonal antibody, which is not specific and takes too long to detect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 125

[0037] Example Synthesis of 125-hydroxyvitamin D3 antigen and preparation of enzyme-linked immunoassay kit

[0038] 1.125-hydroxyvitamin D3 antigen synthesis

[0039] A. Dissolve 100mg bovine serum albumin (BSA) in 10ml pH8.50.05M phosphate buffer;

[0040] B. Dissolve 5mg of 25-hydroxyvitamin D3 in 0.5ml of ethanol, then add 108mg of disuccinimidyl carbonate (pre-dissolved in 400μl of dimethylformamide), and stir the mixture overnight in the dark at room temperature ( Generally 16~22h);

[0041] C. Add 2ml of 0.05M pH10.4 disodium hydrogen phosphate solution containing ethylenediamine and ethylenediamine dihydrochloride to the dissolved bovine serum albumin solution (the concentration of ethylenediamine in the disodium hydrogen phosphate buffer is 10v / v%, ethylenediamine dihydrochloride concentration is 20mg / ml), react at room temperature for 3~4 hours;

[0042] D. After the reaction is completed, add 1 ml of 37% formaldehyde solution to the solution obtained in C, and stir evenly a...

Embodiment 225

[0057] Example 225-Synthesis of Hydroxy Vitamin D3 Antigen and Preparation of Chemiluminescence Immunoassay Kit

[0058] 1.125-hydroxyvitamin D3 antigen synthesis

[0059] A. Dissolve 100mg of ovalbumin in 8ml of pH8.50.05M borate buffer;

[0060] B. Dissolve 35mg of 25-hydroxyvitamin D in 0.5ml of anhydrous pyridine;

[0061] C. Add 200μl of 4-dimethylaminopyridine (42mg) and acetone (200μl) to the dissolved ovalbumin solution, and stir overnight at room temperature in the dark;

[0062] D. Add 108 mg of disuccinimidyl carbonate (pre-dissolved in 400 μl dimethylformamide) to the solution of B, and stir the mixture overnight in the dark at room temperature;

[0063] E. Dissolve 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 1ml of pH8.50.05M borate buffer, and then add dropwise to D and C In the mixed solution, stir overnight in the dark at room temperature;

[0064] F. The final product was dialyzed with 0.01MpH7.4 phosphate buffer for 16 hours, and the dialysat...

Embodiment 31

[0074] Example 31, Synthesis of 25-dihydroxyvitamin D3 antigen and preparation of time-resolved immune kit

[0075] 1.1 Synthesis of 1,2-dihydroxyvitamin D3 antigen

[0076] A. Dissolve 80mg of hemocyanin in 5ml of pH8.50.05M Tris-HCl buffer;

[0077] B. Dissolve 35mg of 1,25-dihydroxyvitamin D in 0.6ml dimethyl sulfoxide; dissolve 3.5mg lysine and 3mg 1-hydroxybenzotriazole in 0.25ml dimethylacetamide, add 16μl N,N'-dicyclohexylcarbodiimide, shake for 1 hour at room temperature, then add 1.5mg 4-dimethylaminopyridine, add the mixture to 1,25-dihydroxyvitamin D3 in dimethyl sulfoxide solution In the medium, the reaction was stirred at room temperature and protected from light for 5 hours, and the product was dried in vacuum;

[0078] C. Add 2ml of glutaraldehyde dropwise to the dissolved hemocyanin solution and mix thoroughly;

[0079] D. Dissolve the product obtained in B in 0.5 ml of dimethyl sulfoxide, add hemocyanin solution dropwise to it, and react in a constant temperature shak...

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Abstract

The invention provides a vitamin D synthetic antigen and a preparation method thereof. The vitamin D synthetic antigen is a conjugate of vitamin D and a protein carrier. The vitamin D is 25-hydroxy vitamin D3 or 1,25-dihydroxy vitamin D3; and the protein carrier is one or more selected from bovine serum albumin, ovalbumin, hemocyanin and human serum albumin. The invention also provides application of the vitamin D synthetic antigen to vitamin D immunological detection. The invention further provides a vitamin D detection kit, which integrates the advantages of existing clinical vitamin D detection methods; and the kit can be applied to all enzyme mark instruments, chemiluminescence instruments and time-resolved analyzers, and has greatly shortened detection time, and sensitivity, accuracy and precision met the detection requirements. The immunosorbent assay kit with strong versatility provided by the invention can realize batch and rapid detection on vitamin D in serum (or plasma).

Description

Technical field [0001] The invention relates to the field of immunology, in particular to a vitamin D synthetic antigen, its preparation method and application. Background technique [0002] Vitamin D is a very important vitamin in the body. Its main role in the body is to regulate the metabolism of calcium and phosphorus. It also has a great relationship with the health of the body. [0003] The main circulating form of vitamin D is 25-hydroxy vitamin D (including 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3), which is the most important vitamin D metabolite and is widely used. When the body lacks vitamin D, it can cause a series of diseases. Among them, 1,25-dihydroxyvitamin D3 has the greatest biological activity. 1,25-dihydroxyvitamin D3 can promote calcium absorption and has other important biological functions. [0004] Due to the importance of vitamin D, the detection of vitamin D content in the body has attracted more and more attention. [0005] Due to the special molecu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K1/113G01N33/82G01N33/577G01N33/543
CPCC07K1/1077C07K14/765C07K14/77C07K14/795C07K19/00G01N33/554G01N33/82
Inventor 杨奇崔建华
Owner BEIJING BOHUI INNOVATION TECH
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