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Anti-human aryl hydrocarbon receptor monoclonal antibody and use thereof

A monoclonal antibody, aryl hydrocarbon receptor technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, instruments, etc.

Active Publication Date: 2019-08-06
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, monoclonal antibodies can only detect one epitope on an antigen

Method used

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  • Anti-human aryl hydrocarbon receptor monoclonal antibody and use thereof
  • Anti-human aryl hydrocarbon receptor monoclonal antibody and use thereof
  • Anti-human aryl hydrocarbon receptor monoclonal antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Preparation of embodiment 1 GST protein and GST-hAhR protein

[0060] I. Expression of GST protein

[0061] Take 10 μL of transformed BL21 competent cells containing the recombinant construction plasmid pGEX4T-1 (Promega) and inoculate them in 20 mL of Amp-resistant LB medium, and activate overnight at 37°C and 180 rpm. Then diluted 1:50 into 1L Amp-resistant LB culture medium, cultured with shaking at 37°C and 180rpm until OD600nm=0.4-0.6 in the logarithmic growth phase, added ITPG with a final concentration of 0.5mM, and induced by shaking at 37°C and 180rpm Cultivate for 3h. Centrifuge at 4500rpm for 10min at 4°C to collect the bacterial liquid precipitate. A total of 2 L of bacterial fluid precipitates were collected.

[0062] II. Purification of GST protein

[0063] (1) Lyse the bacterial liquid precipitate: add 6 times the volume of 1xPBS (30mL) and protease inhibitor with a final concentration of 2nM to the bacterial liquid precipitate collected after centrif...

Embodiment 2

[0071] Example 2 Establishment of Hybridoma Cells and Preparation of Anti-aryl Hydrocarbon Receptor Protein Monoclonal Antibody

[0072] I immune animal

[0073] Seven 6-week-old Balb / c female mice were immunized with the prepared antigen GST-hAhR protein. The numbers of Balb / c female mice are: 1#, 2#, 3#, 4#, 5#, 6# and 7#. Take 280 μL of the purified soluble protein GST-hAhR at a concentration of 0.5 μg / μL, vortex with 1 mL of PBS and 1.2 mL of Freund’s complete adjuvant for 5 min until completely mixed. After fully emulsified, inject 0.48mL subcutaneously in the back of each Balb / c female mouse. After that, Freund's incomplete adjuvant was used instead of complete Freund's adjuvant every two weeks, and other experimental conditions were kept unchanged for the second and third immunizations. One week after the third immunization, blood was collected from the tail of the mice to determine the serum titer (see Table 1).

[0074] Table 1 Determination of serum titer of immu...

Embodiment 3

[0116] The preparation of embodiment 3 monoclonal antibody

[0117] 1. Preparation of Mouse Ascites Containing Monoclonal Antibody

[0118] (l) Six-week-old robust female Balb / c mice were selected and pretreated with 0.2 mL pristine (Sigma) intraperitoneally injected one week in advance.

[0119] (2) Gently blow down the well-grown hybridoma cells, collect them, centrifuge them at 1000 r / min for 3 minutes, resuspend them in sterile NaCl injection, and count them for later use.

[0120] (3) Each mouse was intraperitoneally inoculated with hybridoma cells 5x10 5 indivual.

[0121] (4) Hybridoma cells proliferate in the peritoneal cavity of mice, and produce and secrete monoclonal antibodies. On the 10th to 14th day, the abdomen of the mouse was enlarged, and the ascites was collected.

[0122] (5) Mice were killed by neck dislocation to collect ascites, and 50 μL of 2% Procline300 was added to a 5 mL tube. The ascites sucked out was placed in a 5mL centrifuge tube and mixed...

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Abstract

The invention belongs to the field of biology and environmental detection, and particularly relates to an anti-human aryl hydrocarbon receptor monoclonal antibody, a hybrid tumor generating the monoclonal antibody, a combination comprising the monoclonal antibody, and a using method and application of the monoclonal antibody. The monoclonal antibody can recognize the peptide fragment VCYNPDQIPPENSPL of human aryl hydrocarbon receptors in various cells and can detect human aryl hydrocarbon receptors expressed in situ in cells, the advantage is more favorable for research on the activating process of aryl hydrocarbon receptors and positioning, representation and analysis of in-situ aryl hydrocarbon receptors.

Description

technical field [0001] The invention belongs to the field of biological and environmental detection, and in particular relates to an anti-human aromatic hydrocarbon receptor monoclonal antibody, a hybridoma producing the monoclonal antibody, a composition containing the monoclonal antibody, and a method for using the monoclonal antibody and uses. Background technique [0002] Human aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor. AhR belongs to a newly discovered subfamily of the helix-loop-helix superfamily—a member of the bHLH-PAS subfamily in PAS, and has a basic helix-loop-helix-PAS domain. According to the different functions of each part, AhR can be roughly divided into three functional domains from N-terminus to C-terminus: bHLH domain, PAS domain and transcription activation domain (TAD). The bHLH domain located at the N-terminal of AhR molecules is necessary for protein dimerization and DNA binding, in which the b region determines the s...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N5/20C07K7/08G01N33/577C12R1/91
Inventor 赵斌田文静谢群慧裴新辉胡芹
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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