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Bovine monocyte chemoattractant protein-1 hybridoma cell line, its secreted monoclonal antibody and its application

A technology of hybridoma cell lines and monoclonal antibodies, applied in the direction of anti-cytokine/lymphokine/interferon immunoglobulin, immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problem of natural bovine MCP-1 Poor effect and other problems, to achieve the effect of strong affinity, good specificity, good sensitivity and specificity

Active Publication Date: 2020-02-07
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the anti-bovine MCP-1 (BoMCP-1) antibody prepared in the prior art often encounters great problems when applied to the above-mentioned detection method, especially when it is used to detect natural bovine MCP-1, the effect is not good

Method used

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  • Bovine monocyte chemoattractant protein-1 hybridoma cell line, its secreted monoclonal antibody and its application
  • Bovine monocyte chemoattractant protein-1 hybridoma cell line, its secreted monoclonal antibody and its application
  • Bovine monocyte chemoattractant protein-1 hybridoma cell line, its secreted monoclonal antibody and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1: the acquisition of hybridoma cell line

[0120] Obtaining the hybridoma cell line 4G9 with the preservation number CCTCC NO: C2017281.

[0121] 1. Animal immunization

[0122] The specific immunization procedure is as follows: for the first immunization, 100 μg purified bovine MCP-1 protein fully emulsified with Freund’s complete adjuvant was injected subcutaneously at multiple points in the abdomen, and 100 μg purified bovine MCP-1 protein fully emulsified with Freund’s incomplete adjuvant was injected subcutaneously at multiple points 2 weeks later. The protein was immunized for the second time, and after an interval of 2 weeks, 100 μg of purified protein without adjuvant was injected intraperitoneally for the third immunization, blood was collected 7 days later to determine the serum antibody titer, and mice with higher titer were selected for intraperitoneal booster immunization with 100 μg without adjuvant of purified protein.

[0123] 2. Cell Fusio...

Embodiment 2

[0133] Embodiment 2: Preparation of bovine MCP-1 monoclonal antibody

[0134] 1. Ascites Preparation

[0135] The method of inducing ascites in vivo was carried out according to the conventional method. 10-12 weeks old healthy BALB / c mice were intraperitoneally injected with liquid paraffin 0.3-0.5mL / mouse, and 7-10 days later, the hybridoma cells 4G9 diluted in PBS and cultured to logarithmic phase growth were intraperitoneally inoculated, 5×10 5 After seven days, collect the ascites, centrifuge to remove the precipitate, collect the supernatant, measure the antibody titer by indirect ELISA, aliquot, and store at -70°C.

[0136] 2. Antibody Purification

[0137] The prepared MAb 4G9 ascites was purified by Protein A affinity chromatography.

Embodiment 3

[0138] Example 3: Detection of Monoclonal Antibody Characteristics

[0139] 1. Identification of mAb subclasses

[0140] According to the instructions of the monoclonal antibody subclass kit, the antigen-mediated ELISA method was used. Add 100 μL / well of the cell culture supernatant to the coated ELISA plate, wash at 37°C for 1 hour, wash 3 times with PBST for 5 minutes each time; add 1:1000 diluted goat anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM subclass antibodies 50 μL / well, 37°C for 0.5 h, add two wells of each subclass to each monoclonal antibody, wash 3 times with PBST for 5 min each time; add 1:5000 diluted rabbit anti-sheep enzyme-labeled secondary antibody 50 μL / well Wells were washed 3 times with PBST at 37°C for 15 minutes; 100 μL / well of TMB chromogenic solution was added, and the color was developed at 37°C for 10-15 minutes in the dark, 2M H 2 SO 4 50 μL / well was used to stop the reaction, and the subclass antibody added to the subclass whose color was sig...

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Abstract

The invention provides a hybridoma cell strain 4G9 secreting a bovine monocyte chemoattractant protein 1 (MCP-1) resisting monoclonal antibody as well as a monoclonal antibody MAb 4G9 secreted by same. The monoclonal antibody MAb 4G9 has the advantages of high titer, good specificity and high affinity with natural bovine MCP-1 antigen, and a Western-Blot detection kit, a direct immunofluorescence(DFA) detection kit and a flow cytometry (FCM) detection kit established on the monoclonal antibody MAb 4G9 have good sensitivity and specificity and can be used for detecting recombinant bovine MCP-1, detecting in vitro tissues, researching and identifying the epitope, performing the import and export inspection and quarantine, and performing the non-diagnosis research such as epidemiologic studyand the like.

Description

technical field [0001] The invention relates to a hybridoma cell strain, in particular to a hybridoma cell strain secreting monoclonal antibody against bovine monocyte chemoattractant protein 1 (MCP-1), the secreted monoclonal antibody and application thereof. Background technique [0002] Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC subfamily of chemokines. MCP-1 mainly chemoattracts monocytes, and makes various inflammatory cells, especially monocytes, gather to the lesion, thus playing an important role in body defense, chronic inflammation and anti-tumor. Under the action of specific chemokines, monocytes in the blood migrate and gather in different diseased tissues to play important biological effects, such as phagocytosis and killing pathogenic microorganisms, presenting antigens and secreting various biologically active substances. Studies have shown that the change of MCP-1 expression level is related to the development of infectious diseases, im...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/24G01N33/68C12R1/91
CPCC07K16/24C07K2317/33C07K2317/35C07K2317/92G01N33/6863G01N2333/523
Inventor 焦新安陈祥徐正中夏爱鸿刘泽李昕朱兆成孟闯潘志明殷月兰孙林
Owner YANGZHOU UNIV
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