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17-ketosteroid immunogen, antibody and detection reagent and preparation method thereof

A ketosteroid and immunogen technology, applied in the biological field, can solve the problems of unsuitability for mass use, backward technology, poor stability, etc., and achieve the effects of high-throughput and rapid determination, improved detection efficiency, and improved accuracy

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

AI Technical Summary

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-ketosteroid detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automatic detection reagents

Method used

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  • 17-ketosteroid immunogen, antibody and detection reagent and preparation method thereof
  • 17-ketosteroid immunogen, antibody and detection reagent and preparation method thereof
  • 17-ketosteroid immunogen, antibody and detection reagent and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0061] Example 2: Preparation of Anti-17-Ketosteroid Specific Antibody

[0062] The 17-ketosteroid 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:

[0063] 1. Dilute the above synthesized 17-ketosteroid 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.

[0064] 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.

[0065] 3. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-17-ketosteroid specific antibody with a titer of 1:30000-1:50000.

Embodiment 3

[0066] Embodiment three: the ELISA test of 17-ketosteroid

[0067] The establishment of the ELISA detection standard curve of 1.17-ketosteroids

[0068] (1) Preparation of standard products

[0069] 17-ketosteroid powder (purchased from Sigma) was dissolved in methanol solution to prepare a 1 mg / ml stock solution. The stock solution was sequentially diluted with ELISA buffer to 20.00 mg / L, 10.00 mg / L, 5.00 mg / L, 2.50 mg / L, 1.25 mg / L and 0.00 mg / L standard solutions. Among them, the ELISA buffer contains 50.0mM Tris, 145mM NaCl and 0.25% BSA.

[0070] (2) Utilize the ELISA test method of 17-ketosteroid to prepare standard curve

[0071] Dilute the anti-17-ketosteroid-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; use PBS After the 96-well ELISA plate coated with anti-17-ketosteroid antibody was washed three times, 200 μL / well of 0.5% BSA solution...

Embodiment 4

[0082] Example 4: Preparation of glucose-6-phosphate dehydrogenase-hapten conjugate

[0083] 1. Preparation of glucose-6-phosphate dehydrogenase (G6PDH) solution:

[0084] (1) Accurately weigh 15mg of G6PDH with a specification of 100KU, dissolve it in 12mL containing 72.6mg (0.05M) Tris, 8mgMgCl at room temperature 2 (3.3mM) and 100mgNaCl, the pH of the solution is 9.0, and this step is carried out in beaker C.

[0085] (2) Add 225 mg reduced nicotinamide adenine dinucleotide (NADH), 135 mg glucose-6-phosphate (G-6-P) and 0.75 mL carbitol (Carbitol) to the above beaker C.

[0086] (3) Add 2 mL of dimethylsulfoxide (DMSO) dropwise to the above beaker C.

[0087] 2.17-Activation of ketosteroid derivatives:

[0088] (1) Weigh 10 mg of the above-mentioned 17-ketosteroid derivative in an anhydrous state, and dissolve it in 600 μL of DMF.

[0089] (2) Lower the temperature of the above solution to -2~-8°C.

[0090] (3) Add 3 μL of tributylamine.

[0091] (4) Add 1.5 μL of iso...

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Abstract

The invention discloses a 17-ketosteroid immunogen, antibody and detection reagent and a preparation method thereof. The prepared 17-ketosteroid immunogen is high in immunogenicity, can conduct induction to obtain the high-potency antibody with the 17-ketosteroid resistance characteristic, and has no cross reaction with frequently-seen 62 types of medicine; by means of the 17-ketosteroid detection reagent prepared from the antibody, the content of 17-ketosteroid in biological samples such as urine can be accurately and rapidly confirmed. Compared with an existing detection reagent on the market, the detection reagent has the advantages of being easy and convenient to operate, high in flexibility, high in specificity, accurate in result and the like, can effectively reduce the detection cost of 17-ketosteroid and is beneficial to large-scale clinical application and popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a 17-ketosteroid immunogen, an anti-17-ketosteroid specific antibody and a 17-ketosteroid detection reagent. Background technique [0002] 17-ketosteroids (17-ketosteroids), its structural formula is as shown in formula (Ⅲ): [0003] [0004] 17-ketosteroids refer to corticosteroids and sex hormones that have a ketone group at the 17th carbon position, including androsterone, epiandrosterone, dehydroepiandrosterone, 11-oxyandrosterone, 11-hydroxyandrosterone, etc. Exist in combined form. 17-ketosteroids are mainly metabolites of androgen secreted by the adrenal gland and testis. The 17-ketosteroids in the urine of women and children are mainly derived from the adrenal cortex, and about 2 / 3 of the 17-ketosteroids in the urine of men are derived from the adrenal glands. Cortex, 1 / 3 originates from testis. Urinary 17-ketosteroids can reflect the overall situation of adrenal corticos...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K14/765C07K14/47C07K16/44C07K1/34C07K1/02G01N33/92G01N33/535G01N33/534G01N33/533G01N33/577
CPCC07K14/47C07K14/765C07K14/795C07K16/44G01N33/533G01N33/534G01N33/535G01N33/577G01N33/92
Inventor 虞留明岳育红张顺利梁玉芳王清涛
Owner 苏州博源医疗科技有限公司
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