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Estradiol derivative, immunogen, antibody, enzyme-labeled conjugate, detection reagent and preparation method thereof

A technology of estradiol and derivatives, applied in the fields of estradiol derivatives, enzyme-labeled conjugates, antibodies, detection reagents and their preparations, and immunogens, which can solve the problem of expensive chemiluminescence immunoassay and radioimmunoassay. To solve the problems of radioactive contamination by method and the inability to provide test results quickly, it achieves the effect of easy implementation and popularization, rapid determination and low requirements.

Inactive Publication Date: 2019-01-01
苏州博源医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods have their own defects, such as radioimmunoassay has serious radioactive pollution; enzyme-linked immunoassay can only be performed manually, with large errors; chemiluminescence immunoassay needs to be equipped with expensive special instruments, etc.
Commonly used instrumental analysis and detection methods for estradiol include: gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, high performance liquid chromatography, etc. Although these instrumental analysis methods have high sensitivity, the detection results have high precision and good accuracy. However, there are defects such as cumbersome sample pretreatment, long measurement time, need to be equipped with special instruments and equipment, complex operation process, long detection time for a single sample, and inability to quickly provide test results, so it is not suitable for clinical large-scale samples. test

Method used

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  • Estradiol derivative, immunogen, antibody, enzyme-labeled conjugate, detection reagent and preparation method thereof
  • Estradiol derivative, immunogen, antibody, enzyme-labeled conjugate, detection reagent and preparation method thereof
  • Estradiol derivative, immunogen, antibody, enzyme-labeled conjugate, detection reagent and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Synthesis of Estradiol Derivatives

[0053] The chemical structure of estradiol derivatives is shown in formula (I):

[0054]

[0055] Formula (Ⅰ).

[0056] The synthetic route and preparation steps of above-mentioned estradiol derivative are as follows:

[0057]

[0058] Concrete synthetic steps are as follows:

[0059] a. Synthesis of Compound 3

[0060]

[0061] Weigh 5.0 g (0.018 mol) of compound 1 and 10 g (0.048 mol) of compound 2 and dissolve them in 100 mL of acetone, then add 7.9 g (0.057 mol) of K 2 CO 3 , to prepare a reaction mixture solution, and then the reaction mixture solution was heated to reflux for 12 hours. The solution after the reaction was completed was filtered, and the residue obtained after removing the solvent was purified through a silica gel purification column to obtain 3.1 g of compound 3 as a white solid.

[0062] b. Synthesis of Estradiol Derivatives

[0063]

[0064] Weigh 3.1 g (7.75 mmol) of compound 3, ...

Embodiment 2

[0066] Example 2. Synthesis of Estradiol Immunogens

[0067] The estradiol immunogen in this example is formed by linking the estradiol derivative shown in formula (I) with bovine serum albumin (BSA), and its structural formula is shown in the following formula (II):

[0068]

[0069] Formula (II).

[0070] The concrete steps of the synthetic method of this estradiol immunogen are as follows:

[0071] a. Weigh 4.08 g of potassium dihydrogen phosphate, 6.39 g of disodium hydrogen phosphate, 12.75 g of sodium chloride, and 1.425 g of magnesium chloride, dissolve them in 1.5 L of deionized water, adjust the pH to 8.2, and make buffer solution A;

[0072] b. Weigh 4.5 mg of BSA and dissolve it in 4.5 ml of the above buffer solution A at 4°C to make a carrier solution;

[0073] c. Weigh 4.5 mg of the estradiol derivative represented by the above formula (I), and dissolve it in 450 μl of the above buffer solution A at 4°C to prepare an estradiol derivative solution;

[0074] d...

Embodiment 3

[0076] Example 3. Preparation of anti-estradiol specific antibody

[0077] The estradiol immunogen prepared in Example 2 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:

[0078] a. Dilute the estradiol immunogen represented by the above formula (II) to 3.0 mg / ml with PBS buffer to obtain an antigen solution, then mix 3.0 ml of the antigen solution with Freund's complete adjuvant, and treat the experimental animals give an injection;

[0079] b. After 2 weeks, mix 3.0 ml of the same antigen solution with Freund's incomplete adjuvant, inject once to the above-mentioned experimental animals, and then inject once every 2 weeks, a total of 6 injections;

[0080] c. Take blood from the immunized experimental animals, separate and purify to obtain anti-estradiol specific antibody with a titer of 1:80000.

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Abstract

The invention relates to an estradiol derivative for detecting estradiol in a biological sample and a preparation method of the estradiol derivative. The structural formula of the estradiol derivativeis shown as the following formula (I): (The formula is shown in the description). By using the estradiol derivative, high immunogenicity estradiol immunogen and a corresponding antibody as well as anestradiol enzyme-labeled conjugate are obtained; in addition, a homogenous phase enzyme immunoassay reagent of the estradiol is prepared. The immunogen of the estradiol has high specificity and highimmunogenicity; the prepared anti-estradiol specific antibody has the advantages of high specificity, high titer and capability of well identifying and combining with estradiol; in addition, the prepared anti-estradiol specific antibody has no cross reaction with common 92 kinds of interferents; the homogenous phase enzyme immunoassay reagent containing the anti-estradiol specific antibody and theestradiol enzyme-labeled conjugate can conveniently, rapidly and accurately determine the estradiol content in the biological sample; besides, multiple samples can be simultaneously measured on an automatic biochemical analyzer, so that high throughput and rapid determination of the estradiol are achieved; the preparation method has the advantages of high accuracy and specificity; the accuracy and detection efficiency are greatly improved compared with those of a previous method.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and specifically relates to an estradiol derivative, an immunogen, an antibody, an enzyme-labeled conjugate, a detection reagent and a preparation method thereof. Background technique [0002] The chemical structural formula of estradiol (E2) is shown in the following formula (IV): [0003] [0004] Formula (Ⅳ). [0005] Estradiol is the main component of human estrogen. E2 in women is mainly produced by ovarian follicles, corpus luteum and placenta during pregnancy. 70% of E2 in blood is combined with sex hormone binding globulin (SHBG), 25 % bound to plasma albumin and the rest in free form. There are very few sources of E2 in men, mainly synthesized by testes or metabolites of testosterone. The synthesis of E2 changes periodically, which can promote the formation and development of female reproductive organs, promote the appearance and maintenance of secondary sexual charact...

Claims

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Application Information

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IPC IPC(8): C07J43/00C07K14/765C07K14/77C07K14/795C07K14/47C07K16/44G01N33/531G01N33/558
CPCC07J43/003C07K14/47C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/531G01N33/558
Inventor 虞留明刘天明耿燕周建平
Owner 苏州博源医疗科技有限公司
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