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Hybridoma cell strain, secreted monoclonal antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in anti-cytokine/lymphokine/interferon immunoglobulin, analytical materials, biological material analysis, etc., can solve the problems of poor effect of natural bovine IP-10, etc. Achieve the effect of shortening detection time, good sensitivity and specificity, and strong affinity

Active Publication Date: 2017-03-22
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the anti-bovine IP-10 (BoIP-10) 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 IP-10, the effect is not good

Method used

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  • Hybridoma cell strain, secreted monoclonal antibody and application thereof
  • Hybridoma cell strain, secreted monoclonal antibody and application thereof
  • Hybridoma cell strain, secreted monoclonal antibody and application thereof

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Experimental program
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Embodiment approach

[0111] As an embodiment of the present invention, using HEK293T cells as an expression host, the detection method for detecting bovine IP-10 protein using the indirect immunofluorescence (IFA) detection method of the present invention includes the following steps:

[0112] 1. Transfection

[0113] ① 24 hours before transfection, HEK293T cells were digested and inoculated in a 24-well plate, 2×10 5 cells / well;

[0114] ②Join 1.5 μL into a 1.5 mL finger tube containing Opti-MEM to a total volume of 50 μL;

[0115] ③ Add 1 μg each of recombinant plasmid pVAX1-GFP-BoIP10 and empty vector plasmid pVAX1, and 2 μL of P3000TM to a 1.5 mL finger tube containing Opti-MEM to a total volume of 50 μL;

[0116] ④ add the containing 50 μL of Opti-MEM was added to the wells of the cell plate after standing still for 5 minutes, and placed in an incubator to continue culturing.

[0117] 2. Cytokine detection

[0118] ① Aspirate the culture supernatant of HEK293T (pVAX1-GFP-BoIP10) and H...

Embodiment 1

[0162] The acquisition of embodiment 1 hybridoma cell strain

[0163] Obtained from the hybridoma cell line with the preservation number CCTCC NO: C2016122.

[0164] 1. Animal immunization

[0165] The specific immunization procedure is as follows: for the first immunization, 100 μg of purified bovine IP-10 protein fully emulsified with Freund’s complete adjuvant was injected subcutaneously at multiple points in the abdomen, and 100 μg of purified bovine IP-10 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.

[0166] 2. Cel...

Embodiment 2

[0176] Embodiment 2: Preparation of bovine IP-10 monoclonal antibody

[0177] 1. Ascites Preparation

[0178] 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 2D5 diluted in PBS and cultured to the logarithmic growth phase were inoculated intraperitoneally, 5×105 Cells / only; collect ascites after 7 days, centrifuge to remove precipitate, collect supernatant, measure antibody titer by indirect ELISA, aliquot, and store at -70°C. The monoclonal antibody secreted by the hybridoma cell line CCTCC NO: C2016122 (corresponding to the hybridoma cell 2D5) was designated as MAb 2D5.

[0179] 2. Antibody Purification

[0180] The prepared MAb 2D5 ascites was purified using Protein G affinity chromatography.

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Abstract

The invention provides a hybridoma cell strain 2D5 capable of secreting an anti-cattle-interferon inducing protein-10(IP-10) monoclonal antibody and a secreted monoclonal antibody MAb 2D5. The monoclonal antibody MAb 2D5 has the advantages of high titer, excellent specificity and strong affinity with natural cattle IP-10 antigen. A blocking ELISA detection kit, a Western-Blot detection kit, an indirect immunofluorescence (IFA) detection kit and a flow cytometry (FCM) detection kit which are established on the basis of the monoclonal antibody MAb 2D5 have higher sensitivity and specificity and can be applied to the detection for recombination expression cattle IP-10, the detection for in vitro tissues and the non-diagnosis purpose research, such as epitope identification.

Description

technical field [0001] The invention relates to a hybridoma cell strain, in particular to a hybridoma cell strain secreting anti-bovine interferon-induced protein-10 (IP-10) monoclonal antibody, the secreted monoclonal antibody and application thereof. Background technique [0002] Interferon-inducible protein-10 (IP-10) is a chemokine secreted by antigen-presenting cells stimulated by cytokines and belongs to the CXC chemokine family. IP-10 is a cytokine screened by Luster et al. in 1985 from the gene expression profile of activated U937 cell line. IP-10 can selectively recruit T lymphocytes, natural killer cells and monocytes to the injury site in infectious diseases, autoimmune diseases, allograft rejection, tumors, chronic inflammation and other diseases, playing an important role. Host immune protection. Recent studies have shown that changes in the expression level of IP-10 are associated with infectious diseases, including inflammatory diseases, immune dysfunction a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/24G01N33/68C12R1/91
CPCC07K16/24G01N33/6863G01N2333/46
Inventor 焦新安陈祥徐正中孟闯沈轩云夏爱鸿闵晶晶李昕沈也驰潘志明孙林
Owner YANGZHOU UNIV
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