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Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73

A technology of fucosylation and Golgi protein, applied in the field of medical products, can solve the problems of many residues, inability to apply routine detection, and low sample concentration

Inactive Publication Date: 2007-10-31
BEIJING HOTGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] The current detection methods for Fuc-GP73 (or Golgi protein 73 heterogeneity) are mainly lectin affinity chromatography and mass spectrometry, but these methods cannot be applied to routine detection
At the same time, the traditional lectin affinity chromatography purifies glycosylated proteins, there are many residues in the medium, and the dilution during elution leads to low sample concentration and other problems.

Method used

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  • Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Production of an enzyme-linked immunosorbent assay kit for detecting liver cancer Fuc-GP73 (Golgi protein 73 heterogeneity):

[0092] The composition of the kit (96 persons) includes:

[0093] 1 vial of GP73 negative and positive controls;

[0094] 1 piece of GP73 monoclonal antibody-coated plate (96 wells);

[0095] 1 bottle of horseradish peroxidase (HRP)-labeled GP73 polyclonal antibody, 6ml / bottle;

[0096] 1 bottle of GP73 standard;

[0097] 1 bottle each of substrate solution A and chromogenic solution B, 5ml / bottle;

[0098] 1 bottle of reaction termination solution, 5ml / bottle;

[0099] Fuc-GP73 washing buffer 25ml / bottle

[0100] Fuc-GP73 eluent 15ml / bottle

[0101] Concentrated washing solution (20 times concentrated solution, 20×) 20ml / bottle

[0102] The specific operation is as follows:

[0103] 1. Prepare GP73-negative and positive controls:

[0104] 1) Serum collection: Obtain the serum of healthy normal people and ascites of liver can...

Embodiment 2

[0145]Example 2: Production of a chemiluminescence detection kit for detecting liver cancer fucosyl GP73 (Golgi protein 73 heterogeneity)

[0146] The kit (48 servings) consists of:

[0147] 1 bottle of GP73 negative and positive control;

[0148] 1 piece of GP73 monoclonal antibody-coated plate (96 wells);

[0149] 1 bottle of horseradish peroxidase (HRP)-labeled GP73 polyclonal antibody, 6ml / bottle;

[0150] 1 bottle of GP73 standard

[0151] 1 bottle each of substrate solution A and chromogenic solution B, 5ml / bottle;

[0152] Fuc-GP73 washing buffer 25ml / bottle

[0153] Fuc-GP73 eluent 15ml / bottle

[0154] Concentrated washing solution (20 times concentrated solution, 20×) 20ml / bottle

[0155] The specific operation is as follows:

[0156] 1. Prepare GP73-negative and positive controls:

[0157] 1) Serum collection: Obtain normal human serum and ascites of liver cancer patients from hospitals or blood banks, and store them at -70°C for later use;

[0158] 2) Packi...

Embodiment 3

[0197] Example 3: Preparation and use of prepacked spin columns

[0198] 1. The prepacked spin column is composed of a spin column and a packing medium. The spin column is composed of an upper centrifuge tube and a lower collection tube. The two are set together to form a spin column. Auxiliary reagents include Fuc-GP73 cleaning solution and Fuc-GP73 eluent.

[0199] 2. Preparation of spin column packing materials: Sepharose4B activated by CNBr from Pharmacia and LCA from Sigma were used for coupling according to the following steps:

[0200] (1) Soak 1.5g Sepharose 4B in 1mmol / L HCl until it swells, transfer it into a sand core funnel, and wash it with 300mL 1mmol / L HCl for about 30min;

[0201] (2) Weigh 50mg of LCA, dissolve in 7.5mL coupling buffer (0.1mmol / L NaHCO3, pH8.3, 0.5mol / L NaCl), combine with washed Sepharose 4B, and turn it upside down in a 10mL test tube with stopper Mix well (room temperature, 2h);

[0202] (3) Wash off uncoupled LCA with 10 mL of coupling ...

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Abstract

The invention discloses a prepack eccentric column of fucosido-fucosyl Golgi protein 73 (or Golgi protein 73 heterogeneous body) relative to liver cancer separation, which is characterized by the following: comprising upper separator tube and lower collecting pipe; packing affinity agent of coupling small lentils agglutinine (LCA); assembling a filter cloth on the bottom of the upper separator tube; packing buffer solution in lower collecting pipe; binding small lentils agglutinine (LCA) and sugar chain fucosido-fucosyl Golgi protein 73; centrifuging through adding into eluent; proceeding purify Fuc-GP73 part. This invention discloses a chemoluminescence testing agent case with the eccentric column and enzyme joint immunoassay agent case with the column and preparing and using method. This agent case can test the content of liver cancer fucosido-fucosyl Golgi protein 73, which can provide support for early stage diagnosis.

Description

technical field [0001] The present invention relates to a prepacked spin column for separating liver cancer fucosylated Golgi protein GP73 (or called Golgi protein 73 heterogeneity), and also relates to a chemiluminescence detection kit containing the prepacked spin column The invention relates to an ELISA quantitative detection kit containing the prepacked spin column and a preparation method of the kit, which belong to the technical field of medical products and are used for early diagnosis of liver cancer. Background technique [0002] The main etiology of HCC (hepatocellular carcinoma) is HBV or HCV chronic liver virus infection, and there is generally a long incubation period before the occurrence of liver cancer—that is, the time from infection of liver virus to occurrence of liver cancer. According to the report of "China Medical News" on January 26, 2006 and the introduction of the article in the sixth issue of "Cancer" in 2005, there are about 120 million hepatitis ...

Claims

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Application Information

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IPC IPC(8): C07K1/14G01N33/68G01N33/577
Inventor 林长青
Owner BEIJING HOTGEN BIOTECH CO LTD
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