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Preparation and application of a Golgi protein 73 (gp73) antigen silica-based magnetic bead conjugate

A silicon-based magnetic bead and Golgi technology, which is applied in the field of immunoassay medicine, can solve the problems of short testing time, low cost and low non-specificity, and achieve the effects of low non-specificity, long validity period and good stability.

Active Publication Date: 2016-10-05
北京惠中医疗器械有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to make up for the deficiency of the existing GP73 detection technology, establish a new method for connecting GP73 and silicon-based magnetic beads, and prepare GP73 silicon-based magnetic beads with extremely low non-specificity and high stability. and this linker as one of the components, combined with other components to form a competitive GP73 quantitative detection kit, which has the advantages of high sensitivity, wide linear range, no hook effect, low cost and short test time.

Method used

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  • Preparation and application of a Golgi protein 73 (gp73) antigen silica-based magnetic bead conjugate
  • Preparation and application of a Golgi protein 73 (gp73) antigen silica-based magnetic bead conjugate
  • Preparation and application of a Golgi protein 73 (gp73) antigen silica-based magnetic bead conjugate

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1 Preparation of self-made silica-based magnetic beads

[0049] 1) Fe 3+ Preparation of magnetic particles with a molar concentration of 0.3mol / L in the mixed solution 1: Accurately weigh FeCl 3 ·6H 2 O4 0.54g and FeCl 2 4H 2 O11.93g was dissolved in 500ml of purified water, stirred and reacted for 2 hours under the protection of nitrogen at 55°C, the stirring speed was 400-650rpm, and the pH of the mixed solution was adjusted to be alkaline through concentrated NaOH solution, and the magnetic ferric oxide particles were obtained from the reaction. That is, magnetic particles are referred to as magnetic particles 1 . After the end, wash the magnetic particles with purified water until neutral. Soak the magnetic particles in a certain amount of purified water and mix well, take out some magnetic particle solution and use the drying method (that is, take a certain volume of magnetic particle solution to dry and weigh the dried magnetic particle mass, and divi...

Embodiment 2

[0054] Example 2 Preparation of GP73 antigen silica-based magnetic bead conjugate

[0055] 1) Accurately measure 200 mg of silicon-based magnetic beads, dissolve in 100 ml of 70% ethanol, add 1 ml of ammonia water, and add 3-aminopropyltriethoxysilane (APTES) to make the final concentration 15% (V / V), Mechanically stir the reaction at room temperature for 17 to 18 hours, then use a magnetic plate to precipitate the magnetic beads, remove the supernatant, and wash with purified water for 5 times to obtain magnetic particles with a certain amount of amino groups modified on the surface.

[0056] 2) Dissolve the amino-modified magnetic particles obtained in step 1) in 100ml of 50mM Tris buffer solution with pH=8.0±0.05, add glutaraldehyde to make the concentration 5% (V / V), and react with shaking at room temperature for 1 hour , after the end, the magnetic beads were precipitated with a magnetic plate, the supernatant was removed, and washed 5 times with purified water to obtain ...

Embodiment 3

[0064] Example 3 Preparation of alkaline phosphatase-labeled anti-Golgi protein 73 (GP73) monoclonal antibody working solution and other reagent components

[0065] 1. Preparation of alkaline phosphatase-labeled anti-Golgi protein 73 (GP73) monoclonal antibody working solution

[0066] 1) Weigh 3mg of 2IT, dissolve it in an aqueous solution containing 50mM Tris, 0.1M NaCl and 0.005M EDTA, pH=8.5±0.05 to 10mg / ml; accurately measure 0.5mg of GP73 monoclonal antibody, and make the solution The concentration of the neutral antibody is 3.5mg / ml, and the solvent of the above-mentioned 2IT solution is used as the solvent, which is placed at the bottom of the reaction test tube; 2IT solution with a ratio of 1 / 20 of its volume is added to the test tube containing the 3.5mg / ml GP73 monoclonal antibody solution for activation. Mix well and react at room temperature for 20 minutes. After the end, use molecular sieve chromatography to remove excess 2IT in the solution to obtain activated a...

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Abstract

The invention provides a GP73 antigen silicon-based magnetic bead conjugate as well as a competition method GP73 quantitative determination kit formed by taking the conjugate as one of the components and matching with other components. The kit disclosed by the invention consists of a Golgi protein 73 (GP73) antigen silicon-based magnetic bead conjugate working solution, a Golgi protein 73 (GP73) calibration material, an alkaline phosphatase labeled Golgi protein 73 (GP73) monoclonal antibody working solution, a cleaning solution and a chemiluminescence substrate. Meanwhile, the invention provides a method for preparing the GP73 antigen silicon-based magnetic bead conjugate and a method for preparing each component in the kit. The method comprises the following steps: preparing the GP73 antigen silicon-based magnetic bead conjugate working solution, preparing an enzyme-labeled antibody, preparing the calibration material, preparing the cleaning solution and preparing the chemiluminescence substrate. The kit provided by the invention can be mainly applied to diagnosis of primary hepatic carcinoma and has the advantages of high sensitivity, wide linear range, hook effect avoidance, low cost, short detection time consumption and the like.

Description

technical field [0001] The present invention relates to the field of immunoanalysis medicine, specifically, the present invention provides a preparation method of Golgi Protein-73 (Golgi Protein-73, hereinafter abbreviated as GP73) antigen silicon-based magnetic bead conjugate with high stability and its application in immunoassay Application in diagnostic kits. Background technique [0002] Hepatocellular carcinoma (HCC) is one of the common malignant tumors. Due to its insidious onset, about 2 / 3 of HCC patients are in the middle and advanced stages when they are first diagnosed clinically. According to statistics, the incidence of liver cancer worldwide exceeds 500,000, and the number is increasing year by year. Among them, the number of liver cancer in China will account for about 50% of them, ranking second in the death sequence of malignant tumors. Medical research shows that if liver cancer is detected early and treated in time, the 5-year survival rate of patients ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/531G01N33/54326G01N33/68
Inventor 霍萌萌刘向祎鲁辛辛路晟
Owner 北京惠中医疗器械有限公司
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