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Preparation and application of monoclonal antibody against pancreatic cancer-associated polypeptide dap44

A monoclonal antibody, pancreatic cancer technology, applied in the field of anti-tumor, can solve the problems of false positive and false negative, and achieve the effect of good stability and good specific recognition ability

Active Publication Date: 2018-07-03
陈勇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although surgery is the only effective means for the treatment of pancreatic cancer, only less than 20% of patients can achieve a satisfactory surgical resection and radical effect
However, as an auxiliary diagnostic index, CA19-9 has certain limitations, such as false positives in benign diseases such as pancreatitis and liver cirrhosis and other malignant tumors of the digestive tract; false negatives in patients with Lewis a negative genotype

Method used

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  • Preparation and application of monoclonal antibody against pancreatic cancer-associated polypeptide dap44
  • Preparation and application of monoclonal antibody against pancreatic cancer-associated polypeptide dap44
  • Preparation and application of monoclonal antibody against pancreatic cancer-associated polypeptide dap44

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Screening and sequencing of DAP44 protein

[0023] Serum protein samples were collected from 50 patients with pancreatic cancer before operation and on the 5th day after operation, pretreated to remove albumin and other macromolecules, and protein concentration and quantification. The affinity chromatography column filled with concanavalin A is prepared, the sample is directly loaded on the affinity chromatography column (separation chromatography), and the detection wavelength is 280nm. Due to the lack of glycosylation sites, most proteins in serum do not interact with concanavalin A on the affinity column, and only a few protein regions show a strong affinity for concanavalin A. What remains is the N-terminally glycosylated glycoprotein. Protein samples were efficiently separated and collected by size exclusion chromatography, and MALDI-TOF-MS mass spectrometry was used to analyze and compare the results of serum glycoprotein profiles of pancreatic cancer p...

Embodiment 2

[0024] Example 2 Preparation and Screening of Mouse Monoclonal Antibody

[0025] mouse immunization

[0026] The DAP44 antigenic epitope peptide was synthesized by polypeptide solid-phase chemical synthesis, the sequence is shown in SEQ ID NO: 2, and the end was KLH-coupled. Take 8-week-old healthy BALB / Ac mice, mix and emulsify an appropriate amount of KLH-coupled hapten with Freund's complete adjuvant 1:1, and perform multi-point subcutaneous immunization on the back. Two weeks after the first immunization, the DAP44 protein was mixed with Freund's incomplete adjuvant, and the mice were immunized again in the same way as before. Thereafter, the third and fourth booster immunizations were carried out every 3 weeks. Three days after the fourth immunization, blood was collected from the eye veins of the mice, and mice with higher serum ELISA titers were selected for cell fusion.

[0027] Cell fusion, establishment of hybridoma cell lines

[0028] Spleen cells were taken and...

Embodiment 3

[0044] Example 3 Immunohistochemical Application of Monoclonal Antibody

[0045] The two strains of DAP44 antibodies No.2D1C11 and No.1E8H3 screened above were used to make pathological sections according to conventional methods, and immunohistochemical staining was performed on paraffin sections of human pancreatic cancer tissues. The specific method is as follows: baked slices, 68 ° C, 20 minutes, conventional xylene dewaxing, gradient alcohol dehydration; 3% H 2 o 2 Incubate at room temperature for 10 minutes to block inactivation of endogenous peroxidase; antigen retrieval: put the slices in 0.01M citrate buffer (pH 6.0), boil for 5-10 minutes, and cool naturally; normal goat serum working solution is sealed at room temperature 30min, pour it out without washing; add primary antibody (1:300) and incubate overnight at 4°C refrigerator, wash 3 times with PBS × 5min; add biotin-labeled secondary antibody dropwise, incubate at 37°C for 30min, wash 3 times with PBS × 5min; dro...

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Abstract

The invention analyzes and sieves pancreatic cancer associated polypeptide DTNBP1 Associated Peptide 44 (DAP44) through chromatography high-efficiency separation and mass spectrum proteomics, and discloses an amino acid sequence thereof, and a DAP44 antigen is synthesized by utilizing a polypeptide solid-phase synthesis technology. The DAP44 antigen is taken as an immunogen, monoclonal antibodies for the antigen are prepared by utilizing a hybrid tumor technology, two high-appetency monoclonal antibodies are determined through antibody valence detection, cross pairing and identification of subclass, the antibodies are used for immunohistochemical detection of DAP44, and a foundation is laid for subsequently establishing a DTNBP1 detection kit.

Description

technical field [0001] The invention belongs to the field of anti-tumor technology, and relates to a pancreatic cancer-related polypeptide DAP44, a hybridoma cell line prepared from a polypeptide antigen immunization experiment animal, and a monoclonal antibody produced by the hybridoma cell line. Background technique [0002] Pancreatic cancer is a dangerous malignant tumor of the digestive tract, and its incidence has been on the rise in recent years. Due to the hidden anatomical location of the pancreas, typical clinical symptoms are generally lacking in the early stage of pancreatic cancer. Most patients with pancreatic cancer are already in the advanced stage when they are discovered, and the tumor has invaded or metastasized to surrounding organs, lymph and blood vessels. The survival prognosis is poor, the median survival time is only 3-5 months, and its 5-year survival rate is less than 5%. Although surgery is the only effective means for the treatment of pancreatic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K16/18G01N33/574
CPCC07K14/47C07K16/18G01N33/57438
Inventor 陈勇方城白泉姚雪彪赵宁郭欣吕行
Owner 陈勇
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