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Glycocholic acid immunodetection reagent and preparing method and detecting method thereof

A technology of immunoassay and glycocholic acid, which is applied in the direction of measuring devices, instruments, disease diagnosis, etc., can solve the problems of low accuracy of radioimmunoassay, poor accuracy of test results, poor stability of reagents, and low degree of automation, so as to achieve easy implementation and Promote the use, high-throughput and rapid determination, and realize the effect of full automation

Active Publication Date: 2014-04-30
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

Radioimmunoassay requires professional radioimmunotherapy facilities, which are difficult to carry out in ordinary laboratories, and the accuracy of radioimmunoassay is low, and radioactive rays will also cause great harm to the health of operators, so it is rarely used in the world
The chemiluminescence method has high sensitivity, but the measurement speed is slow, the accuracy of the test results and the stability of the reagent are poor, and it requires expensive special chemiluminescence detection equipment, which is not conducive to the development of routine laboratories and has obvious limitations in clinical application.
Enzyme-linked immunoassay is generally used for semi-quantitative determination, and the operation is cumbersome, the detection time is long, the degree of automation is low, and the repeatability is poor, which is not conducive to wide application in clinical testing.
[0007] At present, there is a lack of glycocholic acid detection reagents with high sensitivity and specificity in the market, especially good quality automatic detection reagents

Method used

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  • Glycocholic acid immunodetection reagent and preparing method and detecting method thereof
  • Glycocholic acid immunodetection reagent and preparing method and detecting method thereof
  • Glycocholic acid immunodetection reagent and preparing method and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Synthesis and Structure Confirmation of Glycocholic Acid Derivatives

[0077] The chemical structure of the glycocholic acid derivative used in the following examples is shown in formula (IV):

[0078]

[0079] Formula (IV)

[0080] The synthetic route of this glycocholic acid derivative is as follows:

[0081]

[0082] Glycocholic Acid Derivatives

[0083] Concrete synthetic steps are as follows:

[0084] Synthesis of compound 3

[0085]

[0086] 1) Weigh 10.0g (24.5mmol) compound 1 cholic acid, dissolve compound 1 in 100mL anhydrous dimethylformamide (DMF), add 7.04g (36.7mmol) carbodiimide hydrochloride ( EDCI), 4.96g (36.7mmol) hydroxybenzotriazole (HOBT) and 10.7mL (61.2mmol) diisopropylethylamine (DIEA), then add 3.07g (24.5mmol) compound 2 glycine and stir for 4 hours , to obtain a synthetic solution;

[0087] 2) Dilute the above synthetic solution with water and then extract with ethyl acetate (EtOAc), wash the organic layer with water ...

Embodiment 2

[0109] Example 2: Synthesis of BSA-glycocholic acid immunogen

[0110] The BSA-glycocholic acid immunogen is composed of bovine serum albumin BSA and -(CH 2 ) n -O-CH 2 The -COO- group is connected. In this example, the synthesis method of the immunogen is described in detail by taking n=2 as an example. The specific steps are as follows:

[0111] 1) Dissolve 20mg of BSA in 5ml of 0.2M, pH8.5 phosphate buffer solution (Phosphate buffer solution, PBS), and place the above solution in beaker A;

[0112] 2) Add the following chemicals into beaker B and stir to dissolve: 20mg glycocholic acid derivative, 0.35ml dimethylformamide (DMF), 0.35ml ethanol, 0.7ml 10mM potassium phosphate buffer solution at pH 5.0. 40mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 5mg N-hydroxysulfosuccinimide (N-hydroxysulfosuccinimide, Sulfo-NHS), stirred and dissolved at room temperature, reacted for 30 minute;

[0113] 3) Add the solution in beaker B to beaker A dropwise to obtain a mixed so...

Embodiment 3

[0116] Example 3: Preparation of anti-glycocholic acid specific antibody

[0117] Inoculate experimental animal rabbits with the BSA-glycocholic acid immunogen prepared above by conventional methods, and take antiserum after booster immunization. The specific steps are as follows:

[0118] Dilute the synthesized BSA-glycocholic acid immunogen to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with Freund's complete adjuvant, and inject the experimental animal rabbit.

[0119] After 2 to 3 weeks, the rabbits were injected once with 1.0 ml of the same antigen solution and Freund's incomplete adjuvant, and then injected once every four weeks, for a total of 4 injections.

[0120] Blood was collected from the above-mentioned experimental animal rabbit, and the anti-glycocholic acid specific antibody was obtained by separation and purification. The titer of the anti-glycocholic acid specific antibody was determined to be 1:30000.

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Abstract

The invention relates to a glycocholic acid detecting reagent and a preparing method and a detecting method thereof, in particular to a glycocholic acid immunodetection reagent and a preparing method and a detecting method thereof. The glycocholic acid immunodetection reagent comprises a glycocholic acid specificity resisting antibody and an indicating reagent, wherein the indicating reagent is used for detecting the glycocholic acid specificity resisting antibody and a glycocholic acid composite; the glycocholic acid specificity resisting antibody is obtained from glycocholic acid immunogen immune animals. The glycocholic acid immunodetection reagent has the benefits that the glycocholic acid immunogen specificity is strong, the immunogenicity is high, and the prepared glycocholic acid specificity resisting antibody has strong specificity and high valence and does not have any cross reaction with 45 common drugs; a homogeneous enzyme immunodetection reagent containing the glycocholic acid specificity resisting antibody can conveniently, rapidly and accurately determine the content of glycocholic acid in a sample and can simultaneously test multiple samples on a fully-automatic biochemical analysis instrument; the high-throughout rapid measurement of the glycocholic acid is realized, the accuracy is high, the specificity is strong, and both the precision and the detection efficiency are greatly improved.

Description

technical field [0001] The invention relates to a glycocholic acid detection reagent and a preparation and detection method thereof, in particular to a glycocholic acid homogeneous enzyme immunological detection reagent and a preparation and detection method thereof. Background technique [0002] The structural formula of Cholylglycine (CG) is shown in (III): [0003] [0004] Formula (III) [0005] Glycocholic acid is one of the conjugated bile acids formed by the combination of bile acid and glycine. It is synthesized by liver cells, enters the intestinal tract with bile, and returns to the liver through the portal vein. When liver cells are damaged, the ability of liver cells to absorb glycocholic acid Therefore, glycocholic acid is one of the sensitive indicators for evaluating the function of liver cells and the circulation of substances in the hepatobiliary system. Studies have shown that the content of glycocholic acid in patients with liver diseases such as cirr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/576G01N33/531
CPCG01N33/532G01N33/576G01N2800/08
Inventor 虞留明李冬陆丽华张曼胡瑜
Owner 苏州博源医疗科技有限公司
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