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Estriol homogeneous enzyme immunoassay reagent, preparation method and detection method

A homogeneous enzyme immunoassay and detection reagent technology, applied to measuring devices, instruments, scientific instruments, etc., to achieve the effects of saving operating time, accurate results, and strong specificity

Active Publication Date: 2018-05-11
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] At present, the commonly used methods for detecting free estriol or total estriol include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunoassay (ELISA), radioimmunoassay ( RIA), time-resolved fluorescent immunoassay (TRFIA) and chemiluminescence immunoassay (CLIA), etc., but these methods have certain limitations in large-scale clinical application.
At present, there is a lack of estriol 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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  • Estriol homogeneous enzyme immunoassay reagent, preparation method and detection method
  • Estriol homogeneous enzyme immunoassay reagent, preparation method and detection method
  • Estriol homogeneous enzyme immunoassay reagent, preparation method and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: Synthesis of Estriol Immunogen

[0052] The estriol immunogen is formed by linking bovine serum albumin (Bovine Serum Albumin, BSA) to the terminal carboxyl group of the estriol derivative represented by formula (II), and the specific steps are as follows:

[0053] 1. Dissolve 200 mg bovine serum albumin in 50 mL 0.2 M, pH 8.5 phosphate buffer;

[0054] 2. Add the following chemicals into a small beaker and stir to dissolve: 200 mg estriol derivatives, 3.5 mL dimethylformamide, 3.5 mL ethanol, 7.0 mL 10mM potassium phosphate buffer, pH 5.0, 200 mg 1- Ethyl-3-(-3-dimethylaminopropyl) carbodiimide, these chemicals were stirred and dissolved at room temperature for 30 min;

[0055] 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 by dialysis to obtain the estriol immunogen.

Embodiment 2

[0056] Example 2: Preparation of anti-estriol specific antibody

[0057] The estriol immunogen prepared in Example 1 was inoculated into experimental animal rabbits by conventional methods, and antiserum was taken after booster immunization. The specific steps were as follows:

[0058] 1. Dilute the above synthesized estriol 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.

[0059] 2. After 2 to 3 weeks, inject 1.0 mL of the same antigen solution and an equal amount of Freund's incomplete adjuvant to the above-mentioned experimental rabbit once, and then inject once every four weeks, for a total of 4 injections.

[0060] 3. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-estriol specific antibody with a titer of 1:30000~1:50000.

Embodiment 3

[0061] Embodiment three: the ELISA test of estriol

[0062] 1. Establishment of ELISA detection standard curve of estriol

[0063] (1) Preparation of standard products

[0064] Estriol 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 into standard solutions of 16.00ng / mL, 8.00ng / mL, 4.00ng / mL, 2.00ng / mL, 1.00ng / mL and 0.00ng / mL. Among them, the ELISA buffer contains 50.0 mM Tris, 145 mM NaCl and 0.25% BSA.

[0065] (2) Utilize the ELISA test method of estriol to prepare standard curve

[0066] The anti-estriol specific antibody prepared in Example 2 was diluted with PBS to a final concentration solution of 1:7500, and 100 μL / hole was coated on a 96-well enzyme-linked plate, and placed at 4°C for 12-24h; After the 96-well enzyme-linked plate coated with anti-estriol antibody was washed three times, 200 μL / well of 0.5% BSA solution was added, and sealed at 4° C....

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Abstract

The invention discloses an estriol homogeneous phase enzyme immunoassay reagent as well as a preparation method and a detection method thereof and belongs to the technical field of immunoassay reagents. An estriol immunogen prepared by the invention is high in immunogenicity, can be induced to obtain a high-titer anti-estriol specific antibody and does not have any cross reaction with 62 common drugs. The estriol immunoassay reagent prepared by the antibody is capable of accurately and rapidly determining the estriol content in biological samples such as urea, blood and the like. Compared with the existing assay reagents in the market as well as detection methods and preparation methods thereof, the detection reagent disclosed by the invention has the advantages of simplicity and convenience in operation, high sensitivity, high specificity, accurate result and the like, is capable of effectively reducing the estriol detection cost and is favorable for large-scale clinical popularization and application.

Description

technical field [0001] The invention belongs to the field of immunoassay reagents, and in particular relates to an estriol homogeneous enzyme immunoassay reagent, a preparation method and a detection method. Background technique [0002] Estriol (E3), its structural formula is shown in formula (Ⅲ): [0003] [0004] Formula (Ⅲ) [0005] Estriol (E3) is one of the important endogenous female hormones. There are free estriol and conjugated estriol in the human body. Generally, the content of estriol in men and non-pregnant women is lower than 2.2 ng / mL, while the content of estriol in pregnant women is generally higher than 31.6 ng / mL. During the pregnancy examination, the development of the fetus can be judged by detecting the content of E3 in the serum or urine of the pregnant woman. The main source of estriol in women of normal pregnancy age is the metabolites of estradiol and estrone, so the content in serum samples is very low; the main source of estriol in pregnant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535G01N33/543
CPCG01N33/535G01N33/543G01N2333/575
Inventor 朱雪明虞留明
Owner 苏州博源医疗科技有限公司
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