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Magnetic particle chemiluminiscence detection kit for determining content of glycocholic acid in human body

A chemiluminescent reagent, the technology of glycocholic acid, which is applied in the direction of chemiluminescence/bioluminescence, and analysis by making materials undergo chemical reactions, can solve complex and cumbersome operations, gas chromatography and mass spectrometry are not suitable for clinical detection, ELISA method low degree of automation

Inactive Publication Date: 2019-03-26
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain defects in these methods. The RIA method is highly polluted, the ELISA method is low in automation, and the operation is complicated and cumbersome. High performance liquid chromatography, gas chromatography, and gas chromatography and mass spectrometry are not suitable for widespread clinical testing.

Method used

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  • Magnetic particle chemiluminiscence detection kit for determining content of glycocholic acid in human body

Examples

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preparation example Construction

[0055] A method for preparing a magnetic particle chemiluminescence detection kit for measuring glycocholic acid content in human body, comprising the following steps:

[0056] Preparation of reagent R1: 1) Prepare buffer according to the content of reagent R1 buffer components, and adjust pH; 2) Prepare fluorescein isothiocyanate-labeled anti-glycocholic acid-BSA monoclonal antibody; 3) Fluorescent isothiocyanate Dilute the anti-glycocholic acid-BSA monoclonal antibody labeled with R1 buffer.

[0057] Preparation of reagent R2: 1) Prepare buffer according to the content of reagent R2 buffer components, adjust pH; 2) prepare alkaline phosphatase-labeled glycocholic acid antigen; 3) use alkaline phosphatase-labeled glycochlic acid antigen with R2 Buffer for dilution.

[0058] Preparation of magnetic separation reagents: 1) preparation of buffer solution according to the content of magnetic particle buffer components; 2) preparation of magnetic particle coated with anti-fluores...

Embodiment 1

[0076] Example 1 A magnetic particle chemiluminescence detection kit for determining the content of glycocholic acid in human body includes R1 reagent, R2 reagent, magnetic separation reagent, calibrator solution series and substrate solution.

[0077] In this example, the R1 reagents include: 1) R1 antibody: fluorescein isothiocyanate (FITC)-labeled anti-glycocholic acid-BSA monoclonal antibody, the concentration is 0.3 μg / ml; 2) buffer: Tris, the concentration 12.04g / L; sodium azide, the concentration is 1.987g / L; sodium chloride, the concentration is 5.79g / L, bovine serum albumin, the concentration is 9.86g / L; 8 aniline-1-naphthalenesulfonate ammonium salt , the concentration is 0.986g / L; the rest is deionized water. The buffer pH of the R1 reagent is preferably 7.0.

[0078] In this embodiment, the R2 reagents include: 1) R2 antibody: alkaline phosphatase-labeled glycocholic acid antigen at a concentration of 0.5 μg / ml; 2) buffer: commercially available AP Conjugate Stabi...

Embodiment 2

[0082] Example 2 Preparation and determination method of magnetic particle chemiluminescence detection kit for measuring glycocholic acid content in human body

[0083] (1) First, add the 50 μl calibrator series of Example 1 (concentrations are 0, 0.33, 1.5, 7.5, 25, 50 ng / ml) and 50 μl reagent R1 and 50 μl reagent R2 of Example 1 respectively into the reaction tube medium, mix and incubate at 37°C for 15 minutes;

[0084] (2) Combine the above reagent series with 25 μl of the magnetic separation reagent of Example 1, and then continue to incubate at 37° C. for 5 min;

[0085] (3) Wash 3 times with cleaning solution to remove unbound antibodies and impurities;

[0086] (4) Add 150 μl of the luminescence substrate solution in Example 1, and use the Leadman self-developed chemiluminescence detector to measure the relative luminescence intensity (RLU) after ALP catalyzes the substrate luminescence. The results are shown in the following table:

[0087] Table 1

[0088] ...

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Abstract

The invention discloses a preparation method of a magnetic particle chemiluminiscence detection kit for determining the content of glycocholic acid in a human body. The kit contains a glycocholic acidR1 reagent, a glycocholic acid R2 reagent, a magnetic separation reagent, a calibration product liquid series and a chemiluminiscence substrate liquid, wherein the glycocholic acid R1 reagent is fluorescein isothiocyanate labeled anti-glycocholic acid-BSA mouse monoclonal antibody diluent; the glycocholic acid R2 reagent is an alkaline phosphatase labeled glycocholic acid antigen diluent; the magnetic separation reagent is an anti-fluorescein isothiocyanate mouse monoclonal antibody coated magnetic particle diluent; glycocholic acid calibration product liquid contains synthetic glycocholic acid and a buffer solution; and the chemiluminiscence substrate liquid is an alkaline phosphatase catalyzed Tris-HCl buffer solution containing dioxane. According to the kit, the signal strength and sensitivity of immunoreactions are greatly improved, a low-content substance can generate very strong chemiluminiscence signals during immune binding, and the relatively accurate, precise, convenient andrapid method is provided for the detection of glycocholic acid.

Description

technical field [0001] The invention relates to the field of medical diagnostic reagents, in particular to a magnetic particle chemiluminescence detection kit for detecting glycocholic acid content in a human body using magnetic particle chemiluminescence technology and antigen-antibody combination technology. Background technique [0002] Glycocholic acid has a molecular weight of 466.3, which is one of the main components of bile acids. Glycocholic acid can be formed after the carboxyl group of glycine and the hydroxyl group at the end of the side chain of cholic acid are combined through peptide bonds. Glycocholic acid is a primary bile acid, which is converted from cholesterol after complex enzymatic reactions in liver cells. [0003] Glycocholic acid is mainly synthesized by liver cells, discharged into the gallbladder through capillaries and bile ducts, and then enters the duodenum with bile to promote fat digestion and absorption. Most of glycocholic acid is reabsorb...

Claims

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Application Information

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IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 王国武李冉
Owner BEIJING LEADMAN BIOCHEM
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