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Monoclonal antibody against GP73 protein, preparation method and application thereof

A technology of monoclonal antibody and Golgi protein, which is applied in biochemical equipment and methods, anti-animal/human immunoglobulin, and analysis by making materials undergo chemical reactions, can solve the problems of heavy labor, lack, and discomfort Quantitative analysis and other issues to achieve high sensitivity, good specificity, and easy quality control

Active Publication Date: 2011-10-26
BEIJING HOTGEN BIOTECH CO LTD
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0009] However, this study used the Western blot method, which is a semi-quantitative analysis, which is too labor-intensive and not suitable for the high-throughput analysis required for large-sample validation studies.
In addition, the method uses a polyclonal antibody detection method, and the method does not establish quantitative standards
[0010] The purpose of the present invention is to solve the current lack of GP73 quantitative detection kit as a means of early diagnosis and detection of liver cancer in clinical practice, and to provide a highly active anti-GP73 monoclonal antibody and an easy-to-operate, accurate and sensitive detection kit and assay In order to observe the expression level of GP73 from the protein level, an effective comprehensive treatment plan can be adopted as soon as possible to prevent the occurrence of liver cirrhosis and liver cancer

Method used

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  • Monoclonal antibody against GP73 protein, preparation method and application thereof
  • Monoclonal antibody against GP73 protein, preparation method and application thereof
  • Monoclonal antibody against GP73 protein, preparation method and application thereof

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preparation example Construction

[0030] In some embodiments of the present invention, the kit further includes a standard product of Golgi protein GP73. In some embodiments of the present invention, the standard product of the GP73 protein is a full-length GP73 protein, and is in a protein protection stable solution. The GP73 standard product can be prepared through recombinant expression by means of genetic engineering, molecular biology and biochemistry. A preparation method comprises: expressing Golgi protein GP73 with Escherichia coli, and performing affinity purification by using the fusion expressed protein tag to obtain a GP73 standard product.

[0031]In some preferred embodiments of the present invention, the above kit may also contain a microtiter plate, horseradish peroxidase (HRP)-labeled polyclonal antibody and auxiliary reagents. Wherein for the enzyme-linked immunoassay kit, the auxiliary reagent may contain the chromogenic substrate tetramethylbenzidine (TMB), and for the chemiluminescence de...

Embodiment 1

[0071] Example 1: Preparation and Characterization of Monoclonal Antibodies of the Present Invention

[0072] 1) Preparation of GP73 monoclonal antibody:

[0073] a) Construction of Golgi protein GP73 recombinant antigen (immunogen):

[0074] The full sequence of the GP73 gene was artificially synthesized according to the gene sequence number AF236056. According to the multiple cloning site of the pET-15b vector, Nde I and BamH I sites were introduced at both ends. The full length of the obtained sequence is shown in figure 1 (SEQ ID NO: 1). Insert the obtained sequence into the prokaryotic expression vector pET-15b, and the pET-15b-GP73 plasmid structure map is shown in figure 2 . The recombinant plasmid was identified by Nde I and BamH I double enzyme digestion, and whether the inserted fragment was checked by 1.2% agarose gel electrophoresis. The identification results are shown in image 3 , where the first lane is M.Takara DL2,000Marker, and the second lane is the pr...

Embodiment 2

[0092] Example 2: Preparation of ELISA Quantitative Detection Kit for Golgi Protein GP73

[0093] The present embodiment prepared a Golgi protein GP73 enzyme-linked immunoassay kit (96 parts) of the present invention, and its composition comprises:

[0094] 5 bottles of Golgi protein GP73 standard;

[0095] 1 piece of GP73 monoclonal antibody-coated plate (96 wells);

[0096] 1 bottle of horseradish peroxidase (HRP)-labeled GP73 polyclonal antibody, 10ml / bottle;

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

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

[0099] 1 bottle of washing buffer (20×concentrated), 50ml / bottle.

[0100] The specific operation is as follows:

[0101] 1. Preparation of Golgi protein GP73 standard:

[0102] 1) The GP73 protein used is the full-length human GP73 protein with His tag artificially synthesized in part 1) of Example 1.

[0103] 2) Packing: Dilute GP73 protein in 2% BSA solu...

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Abstract

The invention provides a monoclonal antibody against GP73 protein, a preparation method and an application thereof. The invention also provides an immunological quantitative detection kit for detecting GP73 in a PBLs sample and a preparation method thereof. The kit in the invention can be used for monitoring the development level of chronic hepatitis and prognosis, and can be used for early diagnosis of liver cancer. By using the kit in the invention to monitor the expression level of the GP73, the prognosis level and probability to cause cirrhosis and liver cancer can be determined for patients, and the most direct support is provided for preventing, diagnosing and curing liver cancer and hepatitis.

Description

technical field [0001] The invention belongs to the field of biotechnology, more specifically, the invention provides a monoclonal antibody against Golgi protein GP73, its preparation method and application. The invention also provides an immunological quantitative detection kit for detecting Golgi protein GP73 in human peripheral blood samples and a preparation method thereof. The kit can be used to quickly and accurately calculate the expression level of Golgi protein GP73 in human peripheral blood. 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 B virus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/577G01N21/76
Inventor 林长青
Owner BEIJING HOTGEN BIOTECH CO LTD
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