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Vanilmandelic acid immune detection reagent and preparation method thereof

A vanillylmandelic acid and reagent technology, applied in the biological field, can solve the problems of instrument quality and working environment interference, cumbersome operation process, and high instrument requirements, and achieve the effects of reducing detection costs, high immunogenicity, and high sensitivity

Inactive Publication Date: 2015-12-23
苏州博源医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The classic VMA detection methods mainly include: visible light spectrophotometry, which is cumbersome to operate and has poor accuracy; gas chromatography, which has relatively high requirements for instruments and complicated operation; high-phase liquid phase-electrochemical method, which is easy to detect. Due to the interference of instrument quality and working environment, the sensitivity and specificity of the determination need to be further improved; the diazo method, this method is cumbersome to operate, has low reliability, and the recovery rate, repeatability, and stability need to be improved
None of the above determination methods can meet the needs of clinical testing of VMA. At present, there is a lack of VMA detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automated testing reagents.

Method used

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  • Vanilmandelic acid immune detection reagent and preparation method thereof
  • Vanilmandelic acid immune detection reagent and preparation method thereof
  • Vanilmandelic acid immune detection reagent and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Synthesis of Embodiment 1 Vanillylmandelic Acid Immunogen

[0060] Vanillylmandelic acid immunogen is composed of bovine serum albumin (BovineSerumAlbumin, BSA) and vanillylmandelic acid represented by formula (II) The specific steps are as follows:

[0061] 1. Dissolve 200mg of bovine serum albumin in 50ml of 0.2M, pH8.5 phosphate buffer;

[0062] 2. Add the following chemicals into a small beaker and stir to dissolve: 200mg of vanillylmandelic acid, 3.5ml of dimethylformamide, 3.5ml of ethanol, 7.0ml of 10mM potassium phosphate buffer at pH 5.0, 200mg of 1-ethyl-3- (-3-Dimethylaminopropyl) carbodiimide, 50mg N-hydroxyl sulfosuccinimide, these chemicals were stirred and dissolved at room temperature for 30 minutes;

[0063] 3. Add the dissolved solution dropwise to the BSA solution, and stir overnight at 2-8° C. to obtain the antigen; purify the synthesized antigen through dialysis to obtain the vanillylmandelic acid immunogen.

Embodiment 2

[0064] Example 2: Preparation of anti-vanillylmandelic acid specific antibody

[0065] The vanillylmandelic acid immunogen prepared in Example 1 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization, and the specific steps were as follows:

[0066] 1. Dilute the above-mentioned synthetic vanillylmandelic acid immunogen to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with an equal amount of Freund's complete adjuvant, and inject the experimental animal rabbit.

[0067] 2. After 2-3 weeks, inject 1.0ml of the same antigen solution and the same amount of Freund's incomplete adjuvant to the above-mentioned experimental animal rabbit once, and then inject once every four weeks, a total of 4 injections.

[0068] 3. Taking blood from the experimental animal rabbit in step 2, separating and purifying to obtain anti-vanillylmandelic acid specific antibody with a tite...

Embodiment 3

[0069] Embodiment three: the ELISA test of vanillylmandelic acid

[0070] 1. The establishment of the ELISA detection standard curve of vanillylmandelic acid

[0071] (1) Preparation of standard products

[0072] Vanillylmandelic acid powder (purchased from Sigma) was dissolved in methanol solution to prepare a 1 mg / ml stock solution. The stock solution was diluted sequentially with ELISA buffer to 16.00mg / L, 8.00mg / L, 4.00mg / L, 2.00mg / L, 1.00mg / L and 0.00mg / L standard solutions. Among them, the ELISA buffer contains 50.0mM Tris, 145mM NaCl and 0.25% BSA.

[0073] (2) Utilize the ELISA test method of vanillylmandelic acid to prepare standard curve

[0074] Dilute the anti-vanillylmandelic acid-specific antibody prepared in Example 2 with PBS to a final concentration solution of 1:8000, coat 100 μL / well on a 96-well enzyme-linked plate, and place it at 4°C for 12-24 hours; After the 96-well enzyme-linked plate coated with anti-vanillylmandelic acid antibody was washed thr...

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Abstract

The invention discloses a vanilmandelic acid immunogen and a synthetic method thereof, and an anti-vanilmandelic acid antibody obtained from the immunogen and a detection reagent, and preparation methods thereof. The prepared vanilmandelic acid immunogen has high immunogenicity, can be induced to obtain the high-titer anti-vanilmandelic acid specific antibody, and moreover, does not has any cross reaction with 62 common drugs; the vanilmandelic acid detection reagent prepared by the antibody can quickly and accurately determine the content of vanilmandelic acid in biological samples such as urine. Compared with conventional detection reagents on markets, the detection reagent has the advantages of simple and convenient operation, high sensitivity, strong specificity, accurate result and the like, also can effectively reduce vanilmandelic acid detection costs, and is conducive to clinical large-scale popularization use.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a vanillylmandelic acid immunogen, an anti-vanillylmandelic acid specific antibody and a vanillylmandelic acid detection reagent. Background technique [0002] Vanillymandelic acid (Vanillymandelic Acid, VMA), its structural formula is shown in formula (II): [0003] [0004] Formula (Ⅱ) [0005] Vanillylmandelic acid, also known as 3-methoxy-4-hydroxymandelic acid (DL-3-Methoxy-4-hydroxymandelicacid, MHMA) or vanillyl mandelic acid, is the terminal substance of the adrenal medullary hormone catecholamines (CA). The end metabolite, CA is almost completely metabolized in the body, a small part of CA is acted by monoamine oxidase to generate p-dihydroxymandelic acid, most of CA is converted into 3-methoxyephrine or Norepinephrine, which is eventually metabolized to 3-methoxy-4-hydroxymandelic acid. The metabolism of CA in the body is very rapid, and its metabolites are mostly excre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/795C07K14/47C07K1/107C07K16/44C07K16/06G01N33/535
CPCC07K14/765C07K1/1077C07K14/47C07K14/795C07K16/06C07K16/44C07K19/00G01N33/535
Inventor 虞留明邹学森王欣茹
Owner 苏州博源医疗科技有限公司
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