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SNCG monoclonal antibody and application thereof

A monoclonal antibody and antibody technology, applied in the field of immunology, can solve the problems of poor specificity, unusability, and high color background of polyclonal antibodies, and achieve improved sensitivity and specificity, high sensitivity and specificity, and less trauma Effect

Active Publication Date: 2009-11-04
BIOCHAIN BEIJING SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, domestic and foreign studies on SNCG are all carried out using SNCG polyclonal antibodies, and polyclonal antibodies have poor specificity, high color background when used in immunohistochemistry, and the quality is not easy to control, so it is difficult to promote in clinical diagnosis.
However, the detection of SNCG gene expression by PCR method has the disadvantage of being easily contaminated and producing false positives, and at the same time cannot accurately reflect the actual level of the protein, which greatly limits the clinical application of SNCG detection.
Existing monoclonal antibody (Jianping Guo et al., Neuronal protein synnuclein gamma predicts poor clinical outcome in breast cancer. "Int. J. Cancer" 121 (6): 1296-305, 2007; Anti-4E9) cannot be used to detect endogenous SNCG protein levels by other immunological methods other than immunohistochemistry, especially not for the detection of SNCG in body fluids

Method used

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  • SNCG monoclonal antibody and application thereof
  • SNCG monoclonal antibody and application thereof
  • SNCG monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The preparation of embodiment 1 anti-human SNCG monoclonal antibody

[0058] 1. Preparation of SNCG antigen

[0059] The 5' end sense primer 5'-GACGGATCCATGGATGTTTTCAAAGAAGGC-3' (SEQ ID NO.1) and the 3' end antisense primer 5'-ATCCTCGAGCTAGTCTCCCCCACTCTG-3' (SEQ ID NO.2) (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.), and BamH I and Xho I restriction sites were introduced at both ends of the primers. The SNCG cDNA fragment was amplified by standard PCR method, and the product length was 393bp.

[0060] The PCR product was ligated with pGEM-T Easy vector (purchased from Promega Company) to form pGEM-T Easy-SNCG plasmid. The pGEM-T Easy-SNCG plasmid was transformed into Escherichia coli JM-109 strain (purchased from Amersham Biosciences) for amplification. The amplified pGEM-T Easy-SNCG plasmid was digested with BamH I and Xho I, then the prokaryotic expression vector pGEX4T-1 was digested with BamH I and Xho I, and the SNCG gene cDNA fragment o...

Embodiment 2

[0064] Embodiment 2 identifies the purity of αSNCG1

[0065] The subclass of αSNCG1 was identified by conventional ELISA method, confirmed to be IgG2a, purified by Pro.A Sepharose-4B column, and its purity and quantification were identified by 12% SDS-PAGE electrophoresis (monoclonal antibody quantification was performed with BSA protein as the standard). see results figure 1 , showing that the antibody is of high purity and free of bands. Similarly, it is also proved that the αSNCG2 antibody has high purity and no bands, and belongs to the subclass IgG1.

Embodiment 3

[0066] Example 3 Determination of the specificity of αSNCG1 and αSNCG2 antibodies

[0067] 1 μg / ml of GST-SNCG, GST-SNCA (Genbank registration number of SNCA: ID6622, GST-SNCA preparation method is the same as that of GST-SNCG), GST-SNCB (Genbank registration number of SNCB: ID 113893, GST-SNCB The preparation method is the same as that of GST-SNCG) to coat the ELISA plate. Add αSNCG1 and αSNCG2 and incubate at room temperature for 1 hour; wash the reaction wells 3 times with 0.05% Tween-20 / PBS, and wash the reaction wells 2 times with PBS. Horseradish peroxidase-labeled goat anti-mouse IgG antibody was added and incubated at room temperature for 1 hour. The reaction wells were washed 3 times with 0.05% Tween-20 / PBS, and 2 times with PBS. Add horseradish peroxidase substrate TMB to the reaction well, after developing color at room temperature for 15 minutes, add stop solution (12.5% ​​H 2 SO 4 ). Measure the absorbance at OD450 with a microplate reader. see results fig...

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Abstract

The invention relates to an SNCG (synuclein gamma) monoclonal antibody and application thereof. The monoclonal antibody is secreted by a hybridoma cell line of which the preservation number is CGMCC No. 2468, or CGMCC No. 2469. The monoclonal antibody has high sensitivity and specificity, can recognize endogenous SNCG protein and be used for various types of immunological detection, can be applied to the level detection of SNCG protein expression in clinical tissue specimens so as to predict the metastasis trend of tumors and the prognosis of patients, can be applied to the detection of SNCG protein in body fluid of suspected or tumor patients so as to help diagnose tumors, can improve the sensitivity and specificity of tumor diagnosis through the combination with reagents detecting other tumor markers, can help to dynamically observe tumor process and provide reference for individualized treatment protocols, and can also be used for related research and medicament development of SNCG.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a monoclonal antibody against SNCG (synucleinγ) or a biologically active fragment derived from the antibody capable of specifically binding to SNCG, and a hybridoma cell line secreting the antibody. The present invention also relates to a test kit comprising the antibody or its biologically active fragment, an antitumor drug and a detection reagent; the application of the antibody or its biologically active fragment in detecting the level of SNCG protein in a sample, and the optional combination of the antibody with detecting other Application of the combination of reagents of tumor markers in auxiliary diagnosis of tumors, monitoring of tumors and / or prognosis judgment of tumor patients. Background technique [0002] The incidence of malignant tumors has continued to rise globally in recent years. Since the onset of malignant tumors is mostly insidious and there are no obvious symptom...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/18G01N33/577A61K39/395A61P35/00
Inventor 寿成超刘彩云吴建孟麟
Owner BIOCHAIN BEIJING SCI & TECH
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