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Anti-napsin A protein monoclonal antibody and its cell line, preparation method and application

A monoclonal antibody, hybridoma cell line technology, applied in the field of biomedical engineering, can solve the problems of time-consuming, labor-intensive, difficult to type, etc.

Active Publication Date: 2021-05-07
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention patent with the application number 201310081206.7 "Lung Cancer Histological Typing Immunohistochemical Multiple Staining Detection Method" provides a combination of Napsin A, TTF-1, and CK5 / 6 antibodies to overcome the existing difficult histological typing of lung cancer, Time-consuming and labor-intensive deficiencies are individually detected by immunohistochemical techniques

Method used

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  • Anti-napsin A protein monoclonal antibody and its cell line, preparation method and application
  • Anti-napsin A protein monoclonal antibody and its cell line, preparation method and application
  • Anti-napsin A protein monoclonal antibody and its cell line, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Polypeptide Synthesis and Chemical Coupling with Carrier Protein

[0027] The Napsin A protein sequence numbered P15391 was selected as a standard sequence from the Uniprot database (http: / / www.uniprot.org), and its sequence is shown in the sequence table SEQID1, by BLAST (https: / / blast.ncbi.nlm. nih.gov / Blast.cgi) tool to compare its sequence differences with other proteins of the same family, and use the Protean module of DNASTAR8.0 software (www.dnastar.com) to conduct secondary structure, antigenicity and surface accessibility analysis.

[0028] The 238th to 268th amino acids of the protein were selected as the antigenic peptide, and a cysteine ​​was added to the C-terminus of the sequence for coupling with the carrier protein KHL to obtain the immunogen. The immunogen consists of antigenic peptide, KLH carrier protein and the disulfide bond between them. The antigenic peptide is named Napsin A-PEP, and its sequence is shown as SEQID2 in the sequence list...

Embodiment 2

[0039] Example 2: Establishment of 21A5 hybridoma cell line

[0040] 1. Immunity

[0041] The immunogen obtained in Example 1 was emulsified with complete Freund's adjuvant (Sigma Company), and immunized with 4-6 week-old female Balb / c mice or ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. ), the abdomen was subcutaneously injected into each mouse at 6 points, and the dose was 60 μg / only. Immunization was boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Company) at a dose of 30 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 50μg / only.

[0042] 2. Cell Fusion

[0043] Aseptically prepare the mouse splenocyte suspension that reaches the immune standard, mix it with mouse myeloma cell sp2 / 0 (ATCC) at a ratio of 5:1, and centrifuge at 1500rpm for 5min. After the ...

Embodiment 3

[0048] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0049] 1. Ascites preparation

[0050] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted about 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0051] 2. Purification of monoclonal antibodies

[0052] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the manual. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

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Abstract

The invention relates to a new pepsin-like aspartic acid protease (Napsin A) monoclonal antibody, a preparation method of the hybridoma cell line 21A5, and the application of the monoclonal antibody in tumor occurrence and prognosis evaluation. The antigen for preparing the antibody is a preferred antigenic peptide, which is chemically synthesized and coupled with KLH carrier protein as an immunogen to immunize mice. The finally obtained antibody belongs to IgG2b subtype, and the sequence encoding its variable region is cloned by gene way to get. The immunohistochemical detection of the finally obtained antibody on a variety of tumor tissues found that the antibody can well identify the occurrence of tumors and can be used for the immunological diagnosis of these tumors.

Description

technical field [0001] The invention relates to the field of biomedical engineering, in particular to an anti-Napsin A protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] Primary lung cancer (hereinafter referred to as lung cancer) is one of the most common malignant tumors in my country. Cancer statistics in China (Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin. 2016, 66(2): 115-32) pointed out that lung cancer is the cancer with the highest incidence rate, and its mortality rate is also in the forefront. In 2015, there were 733,300 new cases of lung cancer (509,300 males, 224,000 females), and 610,200 deaths from lung cancer in my country (432,400 males, 177,800 females) in the same period, accounting for 21.68% of deaths from malignant tumors. [0003] Carrying out lung cancer screening in high-risk groups is beneficial to early dete...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N5/20G01N33/573G01N33/577C12R1/91
CPCC07K16/40G01N33/573G01N33/577
Inventor 胡滨阳杨清海陈惠玲赵普王小亚
Owner FUZHOU MAIXIN BIOTECH CO LTD
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