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Glycocholic-acid immunodetection reagent based on anti-glycocholic acid specific antibody and preparation method thereof

A glycocholic acid and antibody technology, which is applied in the field of immunoassay to achieve the effects of strong specificity, obvious clinical test advantages and high sensitivity

Inactive Publication Date: 2016-04-13
BEIJING HUAXINYUAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the deficiencies in the existing clinical diagnosis of glycocholic acid, the object of the present invention is to provide a fast, efficient, simple and accurate time-resolved immunochromatographic test strip for detecting the content of glycocholic acid in the sample to be tested and its preparation method

Method used

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  • Glycocholic-acid immunodetection reagent based on anti-glycocholic acid specific antibody and preparation method thereof
  • Glycocholic-acid immunodetection reagent based on anti-glycocholic acid specific antibody and preparation method thereof
  • Glycocholic-acid immunodetection reagent based on anti-glycocholic acid specific antibody and preparation method thereof

Examples

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Effect test

Embodiment 1

[0029] Example 1: Preparation and Identification of Glycocholic Acid Derivatives

[0030] The chemical structural formula of the glycocholic acid derivative glycine methyl succinate used in the following examples is shown in formula 4:

[0031]

[0032] The synthetic route of this compound is as follows:

[0033]

[0034] The concrete synthetic steps of this derivative are as follows:

[0035] Synthesis of Compound 2

[0036]

[0037] (1) Dissolve 50.0 g (122.4 mmol) of compound 1 cholic acid in 120 mL of methanol, pass 3 g of hydrogen chloride gas under stirring in an ice bath at 4°C, and heat to reflux for 20 minutes.

[0038] (2) The above synthetic solution was cooled, filtered, and dried to obtain 42.7 g of the product, a white solid, with a yield of 82.6%.

[0039] Synthesis of Compound 3

[0040]

[0041] (1) Weigh 38.1g (90.2mmol) of compound 2 and dissolve it in 400mL of pyridine, add 21.2g (111.2mmol) of p-toluenesulfonyl chloride (TsCl) at 0°C, and s...

Embodiment 2

[0068] Example 2: Preparation of BSA-glycocholic acid derivative conjugates

[0069] The coupling of bovine serum albumin (BSA) and the glycine derivative conjugate in formula 3 can be used as an immunogen. In this embodiment, R is -(CH 2 )n-, taking n=2 as an example to illustrate the synthesis method of the conjugate, using the conjugate to immunize animals to produce specific antibodies against glycocholic acid, the coupling steps are as follows:

[0070] (1) Weigh 5mg of glycocholic acid derivative, dissolve it in 0.1mL dimethylformamide (DMF); weigh 15mg of N-hydroxysuccinimide, dissolve it in 0.2mLDMF; weigh 15mg Dissolve dicyclohexylcarbodiimide (EDC) in 0.2mL DMF; mix the above three solutions and stir at room temperature for 2h to obtain liquid A;

[0071] (2) Weigh 10mg of BSA and dissolve it in 2mL of 0.05M sodium bicarbonate solution, the resulting solution is B solution;

[0072] (3) Add liquid A dropwise to liquid B while stirring at 4°C, and continue to stir f...

Embodiment 3

[0075] Example 3: Preparation of anti-glycocholic acid specific antibody

[0076] The glycocholate immunogen prepared in Example 2 was used to immunize Balb / c mice to prepare anti-glycocholate monoclonal antibody. The specific steps are as follows:

[0077] The glycocholic acid immunogen was diluted to 0.4g / L, emulsified with an equal volume of Freund's complete adjuvant, and then subcutaneously injected into the neck and back of 6-8-week-old Balb / c female mice. The immunization dose was 40 μg / monkey. The second immunization was carried out every two weeks, the immunization dose was halved, and emulsified with Freund's incomplete adjuvant. Thereafter, booster immunization will be given every four weeks, and the dose of immunization will be the same as the second time. After four immunizations, fundus blood was collected for antibody titer detection.

[0078] The anti-glycocholic acid-specific antiserum titer of the immunized mice was measured, and mice with higher antiserum...

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Abstract

The invention discloses a glycocholic acid specific semiantigen, a preparation method for a glycocholic acid specific monoclonal antibody based on the semiantigen, and an immunity chromatography detection method for detecting glycocholic acid. The above specific monoclonal antibody is obtained from a glycocholic-acid-specific-semiantigen-coupled protein immune animal. The immunogen prepared through coupling of the glycocholic acid specific semiantigen and protein keeps a specific active site of glycocholic acid, and the cross reaction rates of the obtained monoclonal antibody with other 8 kinds of bile acids are all smaller than 10%. A time-resolved immunochromatographic test strip prepared by using the above glycocholic acid specific monoclonal antibody is capable of rapidly determining the content of glycocholic acid in a sample, and specificity and accuracy of current glycocholic acid clinic diagnosis are improved.

Description

technical field [0001] The present invention relates to the field of immunoassay. Specifically, it relates to a glycocholic acid hapten, an antibody prepared using the glycocholic acid hapten, and an immunodetection reagent using the prepared antibody. Specifically, it relates to a glycocholic acid time-resolved fluorescent immunoassay strip, and the antibody used in the test strip is a glycocholic acid-specific monoclonal antibody prepared by immunizing mice with a synthetic glycochlic acid immunogen. Background technique [0002] Cholyglycine (CG) is one of the conjugated cholic acids formed by the combination of bile acid and glycine. In liver cells, cholesterol undergoes a complex enzymatic reaction to convert primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CD-CA) cholic acid, chenodeoxycholic acid (CDCA) and Deoxycholic acid (DCA), etc. There are three hydroxyl groups (C3, C7, C12) on the steroid core of cholic acid, and the carboxyl group a...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K16/44C07J41/00G01N33/558
CPCG01N33/558C07J41/0005C07K14/765C07K16/44C07K19/00
Inventor 陈庆全冯婷婷
Owner BEIJING HUAXINYUAN BIOTECH CO LTD
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