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Kit for determining glycocholic acid content in human body and preparation method

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 improving signal strength, strong anti-interference, and improving repeatability and accuracy.

Active Publication Date: 2014-07-23
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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  • Kit for determining glycocholic acid content in human body and preparation method
  • Kit for determining glycocholic acid content in human body and preparation method
  • Kit for determining glycocholic acid content in human body and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0047] 1. Reagent preparation:

[0048] Solution A: Accurately weigh 2mg of glycocholic acid and dissolve it in 100μl DMF reagent; Solution B: prepare 89.4mg / mL NHS solution with DMF; Solution C: prepare 60.2mg / mL DCC (dicyclohexyl Carbodiimide) solution; Solution D: Weigh 16mg of BSA (bovine serum albumin) and dissolve it in 1mL of 0.02mol / L phosphate buffer solution with pH 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. Add 30.7 μL of liquid B and liquid C 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 hours.

[0053] 3. Put the reacti...

Embodiment 2

[0056] Example 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 immunizations (one primary immunization and three booster immunizations), the immune effect was ideal (identified by indirect ELISA method) After immunization, the spleen cells of BalB / c mice whose serum titer is greater than 10,000) were fused with SP2 / 0 myeloma cells (provided by ATCC), and the glycocholic acid monoclonal was obtained through BSA-CG plate screening (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 more ob...

Embodiment 3

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

[0062] 1. Take 1.5g of latex particles (the particles have a core-shell structure, the milk core is polystyrene polymer, the milk shell is composed of styrene, n-butyl acrylate and methacrylic acid copolymer, and the chemical groups on the surface The difference can be selected from one modified by carboxyl, amino, hydroxyl, hydrazide, and chloromethyl; the diameter of latex particles is 100-450nm, purchased from the market, such as the model provided by JSR Company is P0220 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 20ml of carbodiimide (concentration: 0.067mg / ml) and 20ml of N-hydroxysuccinimide (concentration: 0.033mg / ml) to the above solution, and react for 45 minutes.

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

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Abstract

The invention discloses a kit for determining glycocholic acid content in human body. The kit is characterized by comprising a glycocholic acid R1 reagent, a glycocholic acid R2 reagent and a glycocholic acid calibrator, wherein the R2 reagent is prepared by coating glycocholic acid coupled on protein by latex particles and suspending in a special buffer solution. The glycocholic acid is firstly coupled on macro-molecular protein through a chemical bond to form a plurality of sites, while protein relatively high in molecular weight can be better bonded with the latex particles, and protein coupled with the glycocholic acid further coats the surfaces of the latex particles through a chemical cross-linking method. According to the kit disclosed by the invention, the R1 reagent is prepared by adding a glycocholic acid-protein conjugate to the special buffer solution, wherein inorganic salt, a coagulation accelerator, protein and a preservative are added into the special buffer solution. Through the reagent disclosed by the invention, signal strength of immune reaction is greatly improved so that low-content substances can also generate relatively strong turbidity reaction in 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 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 food. 9...

Claims

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Application Information

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IPC IPC(8): G01N21/82G01N33/531
CPCG01N21/82G01N33/577
Inventor 芮双印
Owner ANHUI DAQIAN BIO ENG LIMITED
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