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Vitamin D synthetic antigen, and preparation method and preparation thereof

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

Active Publication Date: 2014-02-19
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 1

[0037] Example 1 Synthesis of 25-Hydroxyvitamin D3 Antigen and Preparation of ELISA Kit

[0038] 1.1 Synthesis of 25-hydroxyvitamin D3 antigen

[0039] A. Dissolve 100mg bovine serum albumin (BSA) in 10ml pH8.5 0.05M phosphate buffer;

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

[0041] C. Add 2ml of 0.05M pH 10.4 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 solution is 10v / v%, the concentration of ethylenediamine dihydrochloride is 20mg / ml), react at room temperature for 3~4 hours;

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

Embodiment 2

[0057] Example 2 Synthesis of 25-hydroxyvitamin D3 antigen and preparation of chemiluminescence immunoassay kit

[0058] 1.1 Synthesis of 25-hydroxyvitamin D3 antigen

[0059] A, 100mg ovalbumin is dissolved in the borate buffer solution of 8ml pH8.5 0.05M;

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

[0061] C. Add 200 μl of a mixture of 4-dimethylaminopyridine (42 mg) and acetone (200 μl) 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 at room temperature in the dark;

[0063] E. Dissolve 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 1 ml of pH8.5 0.05M borate buffer, then add dropwise to D and C In the mixed solution, stir overnight at room temperature in the dark;

[0064] F. Dialyze the final product with 0.01M pH7.4 p...

Embodiment 3

[0074] Example 3 Synthesis of 1,25-dihydroxyvitamin D3 antigen and preparation of time-resolved immunoassay kit

[0075] 1.1 Synthesis of 1,25-dihydroxyvitamin D3 antigen

[0076] A, 80mg hemocyanin is dissolved in the Tris-hydrochloric acid buffer solution of 5ml pH8.50.05M;

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

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

[0079] D. Dissolve the product obtained in B in 0.5ml dimethyl sulfoxide, add hemocyanin solution dropwise, and reac...

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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 synthetic vitamin D antigen, its preparation method and application. Background technique [0002] Vitamin D is a very important vitamin in the body. Its main function in the body is to regulate the metabolism of calcium and phosphorus, and it also has a great relationship with the health of the body. [0003] The major circulating form of vitamin D is 25-hydroxyvitamin D (including 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3), which is the most important vitamin D metabolite and is widely used. A series of diseases can be caused when vitamin D is deficient in the body. Among them, 1,25-dihydroxyvitamin D3 has the highest 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 also attracted more and more attention. [...

Claims

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Application Information

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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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