Preparation method of kit for measuring glycocholic acid content in human body

A glycocholic acid and kit technology, applied in the field of biochemistry, can solve the problems of low value repeatability, low sensitivity, radioactive contamination, etc., and achieve the effects of improved repeatability and accuracy, strong anti-interference, and improved signal strength.

Active Publication Date: 2016-02-03
ANHUI DAQIAN BIO ENG LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently known methods for the determination of glycocholic acid include radioimmunoassay (RIA), chemiluminescence, enzyme-linked immunosorbent assay (ELISA), latex-enhanced turbidimetry, etc., but the steps of radioimmunoassay are cumbersome and the reagents are expensive, requiring Use supporting equipment and there is radioactive contamination
Enzyme-linked immunosorbent assay has long detection time, complicated operation, poor repeatability, and is not suitable for emergency and clinical patients in time for diagnosis.
Although the existing latex-enhanced immunoturbidimetric method is simple and fast, it has low sensitivity and poor repeatability of low values.

Method used

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  • Preparation method of kit for measuring glycocholic acid content in human body
  • Preparation method of kit for measuring glycocholic acid content in human body
  • Preparation method of kit for measuring glycocholic acid content in human body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Preparation of Hepatic Bile Acid-Protein Conjugate

[0047] 1. Reagent preparation:

[0048] Liquid A: Accurately weigh 2 mg of glycocholic acid, and dissolve it in 100 μl of DMF reagent; Liquid B: prepare 89.4 mg / mL NHS solution with DMF; Liquid C: prepare 60.2 mg / mL DCC (dicyclohexylcarbon Diimine) solution; solution D: Weigh 16 mg of BSA (bovine serum albumin), and dissolve it in 1 mL of 0.02 mol / L phosphate buffer solution with a pH of 7.2.

[0049] The phosphate buffers in the examples of the present invention are all selected from commercially available disodium hydrogen phosphate-sodium dihydrogen phosphate or dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer.

[0050] 2. Reagent configuration:

[0051] 1. Take 30.7 μL of liquid B and liquid C respectively, add to 100 μl of liquid A, and stir at room temperature for 90 minutes.

[0052] 2. Add the stirred mixed solution into D solution and mix vigorously, and stir at 4°C for 12 hou...

Embodiment 2

[0056] Embodiment 2 Preparation of glycocholate monoclonal antibody

[0057] 1. Preparation

[0058] BalB / c mice were subcutaneously immunized with KLH protein-coupled CG (BalB / c is a commercially available mouse species). After four times of immunization (one initial immunization and three booster immunizations), the immune effect was ideal (identified by indirect ELISA method) After immunization, the splenocytes of BalB / c mice whose serum titer is greater than 10,000) were fused with SP2 / 0 myeloma cells (provided by ATCC), and monoclonal glycocholic acid was obtained by screening with BSA-CG plates (existing equipment) cell antibodies.

[0059] 2. Screening and identification of hepatobiliic acid monoclonal antibodies

[0060] To identify the small molecule monoclonal antibody, select "BSA-CG coated ELISA plate" to pass the competition ELISA experiment, and the antibody that has a competitive inhibition reaction with free CG is the monoclonal antibody specific to CG. The ...

Embodiment 3

[0061] Example 3 Preparation of hepatic bile acid-protein antibody and latex reactant

[0062] 1. Put 1.5g latex particles (this particle is a core-shell structure, its milk core is a polystyrene polymer, and the milk shell is made up of styrene, n-butyl acrylate and methacrylic acid copolymer, and the chemical groups carried on its surface Different ones can be selected from carboxyl, amino, hydroxyl, hydrazide, and chloromethyl modification; the latex particle diameter range is 100-450nm, which can be purchased from the market, such as the model provided by JSR company is P0220 type carboxyl microspheres) adding PH 5.5, the concentration is in 150ml of 2-morpholinoethanesulfonic acid buffer solution of 80 mmol / L, stirred for 30 minutes;

[0063] 2. Add 20 ml of carbodiimide (concentration: 0.067 mg / ml) and 20 ml of N-hydroxysuccinimide (concentration: 0.033 mg / ml) to the above solution, and react for 45 minutes.

[0064] 3. Centrifuge the above mixture at 15,000 rpm for 15 ...

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Abstract

The invention discloses a preparation method of a kit for measuring glycocholic acid content in the human body. The kit comprises a glycocholic acid R1 reagent, a glycocholic acid R2 reagent and a glycocholic acid calibrator. The glycocholic acid R1 reagent is obtained by sufficiently mixing glycocholic acid-protein conjugate with buffer liquid, the glycocholic acid R2 reagent is obtained by mixing latex particles wrapped by a glycocholic acid-protein antibody with suspension buffer liquid, and the glycocholic acid calibrator is composed of human-derived glycocholic acid and buffer liquid. The preparation method comprises the steps that human plasma containing glycocholic acid, a serum sample and the glycocholic acid-protein conjugate are competitively bound with the glycocholic acid-protein antibody latex particles, the glycocholic acid-protein antibody latex particles bound with the glycocholic acid do not produce turbidity, while, the glycocholic acid-protein antibody latex particles bound with the glycocholic acid-protein conjugate will produce turbidity changes, and quantitative analysis on the glycocholic acid is carried out by measuring the lowering degree of the system turbidity after the reaction. By means of the preparation method, the immunoreaction signal intensity is greatly improved, and even low-content matter can produce strong turbidity reaction during immune binding for detection.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a reagent for measuring the content of glycocholic acid (CG) in a human body by using an immunocompetitive method and a latex enhanced turbidimetric method and a preparation method thereof. Background technique [0002] Serum cholyglycine (CG) is one of the conjugated bile acids in which bile acid and glycine are combined twice. In liver cells, cholesterol is transformed into primary bile acid through complex enzymatic reactions. Among them are cholic acid (CA) and chenodeoxycholic acid (CD-CA). There are three hydroxyl groups (C3, C7, C12) on the steroid nucleus of bile acid, and the hydroxyl group at the end of the side chain is combined with glycine through a peptide bond. CG is synthesized by liver cells, and is discharged into the gallbladder through the capillary and bile ducts, and enters the duodenum along with the bile. Intestines, which aid in the digestion of foo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N21/82
CPCG01N21/82G01N33/577
Inventor 芮双印
Owner ANHUI DAQIAN BIO ENG LIMITED
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