Anti-CBirl hybridoma cell strain and monoclonal antibody produced by same

A hybridoma cell line and monoclonal antibody technology, applied in the field of immunology, can solve the problems of difficulty in obtaining serum or plasma, operator threat, raw material limitation, etc., and achieve the effects of chromosome stability, high titer, and avoidance of objective restrictions

Inactive Publication Date: 2018-07-10
苏州和锐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, human-derived blood products are likely to contain pathogenic infectious substances, which pose a potential threat to the operator. At the same time, a large amount of serum or plasma is required in the process of preparing a positive control, and serum or plasma for some diseases is difficult to obtain.
Therefore, the use of human blood as a positive control, for the preparation of a large number of detection kit products, the raw materials are subject to objective restrictions

Method used

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  • Anti-CBirl hybridoma cell strain and monoclonal antibody produced by same
  • Anti-CBirl hybridoma cell strain and monoclonal antibody produced by same
  • Anti-CBirl hybridoma cell strain and monoclonal antibody produced by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: CBir1 antigenic protein preparation

[0032] 1. Reagents and instruments

[0033] 1) Reagent

[0034] Escherichia coli host bacteria DH5α, BL21(DE3), cloning vector pCR2.1 T-vector, expression plasmid pET28a(+), DNA polymerase rTaq, T4 DNA ligase, DNA polymerase rTaq, LA Taq and restriction endonuclease BamH I, Hind III and EcoR I, BamH I, DL2000 DNA Marker, T4 DNA ligase, low molecular weight standard protein, DNA gel recovery kit, IPTG, etc.

[0035] 2) Instrument

[0036] Ordinary shaker SCS-24; water-proof constant temperature electric heating incubator; Biophotometer spectrophotometer, desktop refrigerated centrifuge Centrifuge 5810R, desktop centrifuge MiniSpin; high-speed refrigerated centrifuge; protein electrophoresis and gel imaging system; PCR instrument; ultrasound Lysis instrument; constant temperature metal bath; HIS protein purification column, etc.

[0037] 2. Experimental method

[0038] 1) Vector construction:

[0039] Design the pr...

Embodiment 2

[0044] Embodiment 2: the preparation of anti-CBir1 hybridoma

[0045] 1. Animal immunization

[0046] The purified CBir1 antigen was mixed with complete Freund's adjuvant in equal volume, emulsified with double syringes, and immunized 6-8 weeks old BALB / c mice by subcutaneous or intraperitoneal injection, with an immunization dose of 50 μg / mouse, with an interval of two weeks Afterwards, the second immunization was carried out, emulsified with incomplete Freund's adjuvant, and the immunization dose was 50 μg per mouse. After two times of immunization, the tail blood was taken to measure the serum titer by serial dilution by ELISA method; according to the results, it was determined whether to boost the immunization. Booster immunization was carried out 3 days before fusion, each mouse was intraperitoneally injected with 100 μg antigen without adjuvant, and cell fusion was carried out 3 days later.

[0047] 2. Cell Fusion

[0048] Pre-warm PEG1500 in a 37°C incubator before f...

Embodiment 3

[0054] Example 3: Preparation of anti-CBirl monoclonal antibody by hybridoma cells with deposit number CCTCC NO: C2016162

[0055] 1. Ascites Preparation

[0056] Male BALB / c mice aged 10-12 weeks were intraperitoneally injected with 0.5ml of liquid paraffin, and one week later, each mouse was intraperitoneally injected with monoclonal cell suspension washed and resuspended in PBS with a 1ml syringe, and the amount of cells used was 5×10 6 / Only. Ascites was collected after ascites accumulated in mice.

[0057] 2. Monoclonal Antibody Purification

[0058] The ascites was centrifuged at 10,000 rpm for 10 minutes at 4°C to remove lipids. After centrifugation, the supernatant was aspirated and filtered with a 0.45 μm membrane. protein G was purified. The concentration of the purified monoclonal antibody was measured, subpackaged, and frozen at -20°C.

[0059] 3: ELISA method to identify monoclonal antibody subclasses

[0060] Dilute coated goat anti-mouse IgG (Zhongshan Ji...

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Abstract

The invention discloses a hybridoma cell strain having the preservation number of CCTCC NO: C2016162 and an anti-CBirl monoclonal antibody produced by the same. The invention also relates to an engineered antibody on the basis of the hybridoma cell strain and application of the engineered antibody in immunoassay tools. The engineered antibody can be used as a positive control for human blood.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to the preparation of a hybridoma cell line for producing a monoclonal antibody secreting anti-CBir1, and the application of the CBir1 chimeric antibody prepared based on the hybridoma cell line in the detection of inflammatory bowel disease. Background technique [0002] Inflammatory bowel disease (IBD) is a chronic non-specific intestinal inflammatory disease, including ulcerative colitis (UC) and Crohn's disease (CD), in the past 10 years In my country, the incidence rate is gradually increasing. The clinical manifestations of IBD are complex and diverse, not only with gastrointestinal symptoms, but also with extraintestinal manifestations. Since the symptoms are non-specific enteritis manifestations such as abdominal pain, diarrhea, and blood in the stool, it is difficult to differentially diagnose CD and UC, as well as other intestinal chronic diseases such as IBD and tuberc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/68G01N33/577C12R1/91
CPCC07K16/12G01N33/577G01N33/6893G01N2333/195G01N2800/065
Inventor 陈菲周鹏飞霍如松
Owner 苏州和锐生物科技有限公司
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