Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid

A technology for a diagnostic reagent and a glycocholidase conjugate, which is applied in the field of biomedicine, can solve the problems of unfavorable clinical tests, poor accuracy of test results and reagent stability, complicated operation, etc., and achieves a simple and easy-to-control preparation process and high-efficiency reagents. Accurate and sensitive, simple operation effect

Inactive Publication Date: 2017-02-15
李松羊
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 reagents 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.
[0006] 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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  • Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid
  • Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid
  • Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid

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Embodiment 1

[0042] A preparation method of glycocholic acid homogeneous enzyme immunodiagnostic reagent:

[0043] (1) prepare glycocholic acid antibody solution,

[0044] Add glycocholic acid antibody to Tris-HCl buffer solution with pH 7.4, add 6-phosphate glucose, NADP, BSA, sodium azide, and mix well to obtain glycocholic acid antibody solution. The concentration of the glycocholic acid antibody in the solution is 0.05 mg / ml, and the mass percent content of sodium azide is 0.1%;

[0045](2) preparing a glycocholidase conjugate solution,

[0046] Add glycocholic acid to MES buffer, add carboxyl activator EDC-NHS for carboxyl activation, carboxyl activation temperature is 0°C, time is 5min, pH is 5.5, then add 6 -Phosphoglucose dehydrogenase carries out condensation reaction to obtain glycocholidase conjugate crude product, the mass ratio of 6-phosphoglucose dehydrogenase and glycocholic acid is 1:1000, the glycocholidase conjugate crude product is at pH The glycocholate enzyme conjug...

Embodiment 2

[0050] A preparation method of glycocholic acid homogeneous enzyme immunodiagnostic reagent:

[0051] (1) prepare glycocholic acid antibody solution,

[0052] Add the glycocholic acid antibody to the Tris-HCl buffer solution with a pH of 8.0, add 6-phosphate glucose, NADP, sucrose, and sodium azide, and mix evenly to obtain the glycocholic acid antibody solution. Among them, glycocholic acid The concentration of the antibody is 0.2mg / ml, and the mass percentage content of sodium azide is 0.2%;

[0053] (2) preparing a glycocholidase conjugate solution,

[0054] Add glycocholic acid to MES buffer, add carboxyl activator EDC-NHS for carboxyl activation, carboxyl activation temperature is 10°C, time is 15min, pH is 6.0, then add 6 -Phosphoglucose dehydrogenase carries out condensation reaction to obtain glycocholidase conjugate crude product, wherein, the mass ratio of 6-phosphoglucose dehydrogenase and glycocholic acid is 1:10, and the glycocholidase conjugate crude product is...

Embodiment 3

[0058] A preparation method of glycocholic acid homogeneous enzyme immunodiagnostic reagent:

[0059] (1) prepare glycocholic acid antibody solution,

[0060] Add the glycocholic acid antibody to the Tris-HCl buffer solution with a pH of 8.5, add 6-phosphate glucose, NADP, mannose, and sodium azide, and mix evenly to obtain the glycocholic acid antibody solution. The concentration of the acid antibody is 5mg / ml, and the mass percent content of sodium azide is 0.3%;

[0061] (2) preparing a glycocholidase conjugate solution,

[0062] Add glycocholic acid to MES buffer, add carboxyl activator EDC-NHS for carboxyl activation, carboxyl activation temperature is 20°C, time is 25min, pH is 6.5, then add 6 -Phosphoglucose dehydrogenase carries out condensation reaction to obtain glycocholidase conjugate crude product, wherein, the mass ratio of 6-phosphoglucose dehydrogenase and glycocholic acid is 1:1, and the glycocholate enzyme conjugate crude product is in The glycocholidase c...

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Abstract

The invention relates to a preparation method for a homogeneous enzyme immunodiagnosis reagent used for glycocholic acid, belonging to the technical field of biological medicine. The preparation method comprises the following steps: preparation of a glycocholic acid antibody solution; preparation of a glycocholic acid-enzyme conjugate solution; and preparation of a glycocholic acid calibrating substance. The glycocholic acid-enzyme conjugate solution is prepared by adding glycocholic acid into a MES buffer solution, adding a carboxyl activator for carboxyl activation, then adding 6-phosphogluconate dehydrogenase for a condensation reaction so as to obtain a crude glycocholic acid-enzyme conjugate product, carrying out dialysis and purification, adding the treated crude glycocholic acid-enzyme conjugate product into a Tris-HCl buffer solution, adding an auxiliary reagent and carrying out uniform mixing so as to obtain the glycocholic acid-enzyme conjugate solution. The homogeneous enzyme immunodiagnosis reagent for glycocholic acid prepared by using the method is safe, rapid, highly efficient and sensitive and can accurately detect the content of glycocholic acid in a to-be-detected sample.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a preparation method of a glycocholic acid homogeneous enzyme immunodiagnostic reagent. Background technique [0002] Glycocholic acid is one of the conjugated cholic acids formed by the combination of cholic acid and glycine in serum, and its molecular structure is as follows: [0003] [0004] Glycocholic acid is synthesized by liver cells, enters the intestinal tract with bile, and returns to the liver through the portal vein. When the liver cells are damaged, the ability of the liver cells to absorb Glycocholic acid decreases, resulting in an increase in the blood content. Acute hepatitis, chronic hepatitis, liver cirrhosis, cholelithiasis with jaundice, bile duct and gallbladder excretory dysfunction, obstructive liver disease, intestinal-hepatic circulation disorder, liver cancer will all cause glycocholic acid to increase Glycocholic acid is also an important indicato...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N21/31
CPCG01N33/531G01N21/31
Inventor 李松羊
Owner 李松羊
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