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Hybridoma cell strain capable of secreting anti-Spondin1 monoclonal antibody as well as monoclonal antibody and application of monoclonal antibody

A technology of hybridoma cell line and monoclonal antibody, which is applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, analytical materials, anti-animal/human immunoglobulin, etc., which can solve the problem of poor specificity of antibodies , Sensitivity effects, inability to distinguish Spondin1 highly homologous family proteins, etc., to achieve strong specificity and high sensitivity

Active Publication Date: 2020-12-11
苏州仁端生物医药科技有限公司
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] Most of the existing detection kits are R-Spondin1 kits, and polyclonal antibodies are mostly used in the system; the antibody has poor specificity and cannot distinguish Spondin1 highly homologous family proteins. This limits its clinical utility for Spondin1 detection, reduces the specificity of sample detection, and also has a certain impact on sensitivity

Method used

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  • Hybridoma cell strain capable of secreting anti-Spondin1 monoclonal antibody as well as monoclonal antibody and application of monoclonal antibody
  • Hybridoma cell strain capable of secreting anti-Spondin1 monoclonal antibody as well as monoclonal antibody and application of monoclonal antibody
  • Hybridoma cell strain capable of secreting anti-Spondin1 monoclonal antibody as well as monoclonal antibody and application of monoclonal antibody

Examples

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Effect test

Embodiment 1

[0022] Embodiment 1, recombinant protein expression and purification of Spondin1

[0023] After extracting RNA from ovarian cancer tissue, the cDNA obtained by RT-PCR with random primers was used as a template, and primers were designed to clone the Spondin1 gene. The specific primers were: Spondin1-F: 5'-TGTTCATCCTTGTTA-3' (SEQ ID NO.1); Spondin1-R: 5'-CGGGGACAGCCTCAT-3' (SEQ ID NO. 2). The amplified Spondin1 gene was inserted into the pcDNA3.1 vector containing 6×His tag to obtain Spondin1-pcDNA3.1. Then Spondin1-pcDNA3.1 was transformed into DH5α, positive clones were picked and cultured in large quantities, and the recombinant plasmid Spondin1-pcDNA3.1 was extracted with a high-purity plasmid extraction kit. The recombinant plasmid was transferred into 293T cells, and the pcDNA3.1 empty vector was transfected at the same time as a negative control. 2 Cultivate for 72 hours under the condition, collect the supernatant, and filter the supernatant with a 0.22 μm filter memb...

Embodiment 2

[0025] Embodiment 2, the preparation of Spondin1 monoclonal antibody hybridoma cell line

[0026] Mouse immunization: 3 batches of BALB / c mice were immunized with Spondin1 protein, 3 mice each time. Each mouse was immunized with 50 μg antigen each time. For the first immunization, Spondin1 antigen was mixed with Freund's complete adjuvant 1:1, and then Spondin1 was mixed with Freund's incomplete adjuvant 1:1, immunized once every two weeks, a total of 3 immunizations, and the tail was docked ten days after the third immunization Blood collection, detection of serum titer.

[0027] Hybridoma cell fusion: After detecting that the titer of mouse serum reaches 1:100000, use Spondin1 antigen (without adjuvant) to boost intraperitoneal immunization (30 μg), and aseptically collect spleen cells and SP2 / 0 mouse bone marrow from immunized mice 3 days later Tumor cells were mixed in a 50mL centrifuge tube at a ratio of 4:1, washed twice with medium, discarded the supernatant, and then...

Embodiment 3

[0029] Example 3, Spondin1 monoclonal antibody preparation, purification, titer detection and specificity analysis

[0030]Monoclonal antibody ascites preparation: Culture the 3 screened hybridoma cell lines in culture medium to 90% density, collect the cells, and dilute the cells to 3×10 6 individual / mL. After 10-12 weeks of adaptive feeding of BALB / c mice for one week, intraperitoneal injection of immunosuppressant liquid paraffin, 0.5mL / mouse, 7 days later, the abdomen was inoculated with hybridoma cells that stably secreted antibodies and were in good condition, about (1-2) ×10 6 One per mouse, after 7-10 days, the abdomen of the mice was inflated, the ascites was extracted, and the supernatant of the ascites was collected.

[0031] Antibody purification: Collect mouse ascites, filter the solution with a 0.45 μm filter, add solid ammonium sulfate with a final concentration of 50%, stir evenly at 4°C, let stand for 1 hour, collect the precipitate at 12000 rpm, and redisso...

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Abstract

The invention provides hybridoma cell strains capable of secreting anti-Spondin1 monoclonal antibody, and the preservation numbers of the hybridoma cell strains are CCTCC NO: C202093 and CCTCC NO: C202094 respectively. The invention also provides a monoclonal antibody secreted by the hybridoma cell strain secreting the anti-Spondin1 monoclonal antibody. The invention provides a test kit containingthe monoclonal antibody. The hybridoma cell strains capable of secreting anti-Spondin1 monoclonal antibody are obtained, two monoclonal antibody aiming at different epitopes of the Spondin1 are obtained, the specificity is high, the sensitivity is high, the content of the human bottom plate reactive protein Spondin1 in body fluid and tissue can be detected through pairing, and the invention is applied to detection of Spondin1 protein overexpression or diagnosis of products of diseases characterized by abnormal expression of Spondin1 protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a Spondin1 monoclonal antibody hybridoma cell line. Background technique [0002] Extracellular matrix substrate-response protein 1, or Spondin1, is a cell adhesion protein encoded by the SPON1 gene. It was originally isolated from the embryonic floor plate of vertebrates. Promotes vascular smooth muscle cell proliferation and inhibits angiogenesis. Spondin1 protein contains 807 amino acids, molecular weight is 90.97Kda, contains several disulfide bonds, and is a typical secreted extracellular matrix glycoprotein. [0003] Studies have found that Spondin1 is expressed in many tissues, such as ovary, embryonic growth plate cartilage, periodontal tissue, and osteoarthritis cartilage. With the deepening of research on the pathogenesis of tumors, it is found that Spondin1 plays a very important role in the occurrence, development, invasion and metastasis of tumors. For example, Spondin...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/30G01N33/543G01N33/574G01N33/577G01N33/58
CPCC07K16/3069C07K2317/35G01N33/54326G01N33/57449G01N33/57488G01N33/577G01N33/581
Inventor 庄光磊臧荣余殷霞
Owner 苏州仁端生物医药科技有限公司
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