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17-hydroxycorticosteroid immunodetection reagent and preparation method thereof

A technology of hydroxycorticosteroids and reagents, applied in the biological field, can solve problems such as unsuitable for large-scale use, backward technology, poor stability, etc., and achieve the effects of being beneficial to clinical promotion and use, reducing detection costs, and strong binding force

Inactive Publication Date: 2015-11-25
苏州博源医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is relatively backward in technology and poor in stability, and is no longer suitable for clinical use in large quantities.
At present, there is a lack of 17-hydroxycorticosteroid detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automatic test reagents

Method used

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  • 17-hydroxycorticosteroid immunodetection reagent and preparation method thereof
  • 17-hydroxycorticosteroid immunodetection reagent and preparation method thereof
  • 17-hydroxycorticosteroid immunodetection reagent and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1 7

[0059] Synthesis of embodiment one 17-hydroxycorticosteroid immunogen

[0060] The 17-hydroxycorticosteroid immunogen is composed of bovine serum albumin (BovineSerumAlbumin, BSA) and 17-hydroxycorticosteroid derivatives 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 17-hydroxycorticosteroid derivatives, 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 to the BSA solution dropwise, and stir overnight at 2-8°C to obtain the antigen; purify the synthesized antigen by dialysis to obtain the 17-hydroxycorticosteroid immunogen...

Embodiment 2

[0064] Example 2: Preparation of Anti-17-Hydroxycorticosteroid Specific Antibody

[0065] The 17-hydroxycorticosteroid immunogen prepared in Example 1 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:

[0066] 1. Dilute the above-mentioned synthetic 17-hydroxycorticosteroid 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. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-17-hydroxycorticosteroid specific antibody with a tit...

Embodiment 3

[0069] Embodiment three: the ELISA test of 17-hydroxycorticosteroid

[0070] 1.17-Hydroxycorticosteroid ELISA detection standard curve establishment

[0071] (1) Preparation of standard products

[0072] 17-Hydroxycorticosteroid powder (purchased from Sigma Company) was dissolved in methanol solution to prepare a 1000 μmol / L stock solution. The stock solution was sequentially diluted with ELISA buffer to 80.00 μmol / L, 40.00 μmol / L, 20.00 μmol / L, 10.00 μmol / L, 5.00 μmol / L and 0.00 μmol / 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 17-hydroxycorticosteroid to prepare standard curve

[0074] Dilute the anti-17-hydroxycorticosteroid-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 12-24h; After washing the 96-well enzyme-linked plate coated with anti-17-hydrox...

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Abstract

The invention discloses 17-hydroxycorticosteroid immunogen and a synthesizing method thereof, and a 17-hydroxycorticosteroid antibody obtained through the 17-hydroxycorticosteroid immunogen, a detecting reagent and a preparation method of the 17-hydroxycorticosteroid detecting reagent. The prepared 17-hydroxycorticosteroid immunogen is high in immunogenicity, the high-potency 17-hydroxycorticosteroid specific antibody can be obtained through induction, and no cross reaction happens to the 17-hydroxycorticosteroid specific antibody and 62common drugs. According to the 17-hydroxycorticosteroid detecting reagent prepared through the antibody, the content of 17-hydroxycorticosteroid in urine and other biological samples can be accurately and fast determined. Compared with existing detecting reagents on the market, the detecting reagent has the advantages of being easy and convenient to operate, high in sensitivity, high in specificity, accurate in result and the like, the detecting cost of 17-hydroxycorticosteroid can be effectively reduced, and clinical large-scale use and popularization are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a 17-hydroxycorticosteroid immunogen, an anti-17-hydroxycorticosteroid specific antibody and a 17-hydroxycorticosteroid detection reagent. Background technique [0002] 17-hydroxycorticosteroids (17-hydroxycorticosteroids), its structural formula is shown in formula (Ⅲ): [0003] [0004] Formula (Ⅲ) [0005] 17-Hydroxycorticosteroids refer to adrenocortical hormones and sex hormones with a hydroxyl group at the 17th carbon position, mainly cortisol, corticosteroids and their metabolites secreted by the adrenal cortex, which are mainly excreted by the kidneys. Therefore, the determination of 17-hydroxycorticosteroid in urine is a reliable indicator to reflect the function of adrenal cortex. Decreased 17-hydroxycorticosteroid levels are common in primary or secondary adrenal insufficiency, hypopituitarism, bilateral adrenalectomy, malnutrition, liver cirrhosis, tuberculosis, and cer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/577
CPCG01N33/53G01N33/577
Inventor 虞留明肖九长赵冬
Owner 苏州博源医疗科技有限公司
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