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Hybridoma cell strain 17C8 and monoclonal antibody generated therefrom used in Brucella detection

A technology of hybridoma cell line and monoclonal antibody, applied in the direction of microorganisms, measuring devices, anti-bacterial immunoglobulin, etc., can solve the problem of low sensitivity and achieve the effect of broad application prospects

Inactive Publication Date: 2012-03-28
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The downside of serological methods is that it is less sensitive and prone to cross-reactivity with Vibrio cholerae, Yersinia enterocolitica, EHEC, Salmonella, Shigella flexneri, etc.

Method used

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  • Hybridoma cell strain 17C8 and monoclonal antibody generated therefrom used in Brucella detection
  • Hybridoma cell strain 17C8 and monoclonal antibody generated therefrom used in Brucella detection
  • Hybridoma cell strain 17C8 and monoclonal antibody generated therefrom used in Brucella detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, the acquisition of hybridoma cell line 17C8 CGMCC No.4971 and the monoclonal antibody it produces

[0030] 1. Strain culture

[0031] Take -80°C preserved strain - Brucella melitensis 16M (referred to as 16M, purchased from the Institute for the Control of Biological Products), and inoculate it in TSB medium (purchased from Bio-Mérieux) at a ratio of 1:100. 37°C, 200rpm shaking culture for 48h. Pick a small amount with an inoculation loop, streak on a TSA plate (purchased from Beijing Xinjingke Biotechnology Co., Ltd.), and culture at 37°C for 72 hours, pick a single colony and inoculate it into 5mL TSB medium, shake at 37°C and 200rpm Cultivate for 48h. Transfer to 200mL TSB medium at a ratio of 1:100, and culture to mid-logarithmic phase. Formaldehyde solution with a final concentration of 4% (volume percentage concentration) was added to the bacterial solution, and the bacteria were inactivated by acting at room temperature for 10 minutes. Centrifuge t...

Embodiment 2

[0079] Embodiment 2, sample detection example

[0080] The prepared monoclonal antibody secreted by the hybridoma cell line 17C8 was used to coat the enzyme-linked plate, and the positive control was dissolved in the serum of a healthy person with inactivated Brucella melis 16M, so that the final concentration reached 10 8 CFU / mL, negative control with healthy human serum. 20 brucellosis patient sera were detected by ELISA method, and the specific method included the following steps:

[0081] (1) Coating: Monoclonal antibody produced by hybridoma cell line 17C8 CGMCC No.4971, diluted 1:20000, added 100 μl to each well, overnight at 4°C, washed 3 times, and patted dry.

[0082] (2) Blocking: block the coated enzyme-linked plate with blocking solution, 150 μl / well, and act at 37°C for 2 hours;

[0083] (3) Washing: wash 3 times and pat dry;

[0084] (4) Add samples to be tested: brucellosis patient serum is diluted 1:50, add 100 μl to each well, and set positive and negative ...

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Abstract

The invention provides a hybridoma cell strain 17C8 CGMCC No. 4971 obtained from immune mice with Brucella whole cell bacteria as an antigen, and a monoclonal antibody generated from the hybridoma cell strain 17C8. The preparation method of the monoclonal antibody comprises steps that: (1) the Brucella whole cell bacteria antigen is adopted as an immunogen to immunize an animal; (2) spleen cells of the immune animal are separated, and are fused with myeloma cells, such that hybridoma cells are obtained; (3) the hybridoma cells are screened and cultured; and (4) monoclonal antibodies are separated and purified from cell culture fluid or ascitic fluid of an animal vaccinated with the hybridoma cells. As a result of experiments, the monoclonal antibody generated from the hybridoma cell strain 17C8 CGMCC No. 4971 can subject to reactions with all Brucella strains (strains and vaccine strains), and has high specificity. Therefore, the monoclonal antibody can be used in Brucella detections. According to the characteristics of the monoclonal antibody strain provided by the invention, methods for detecting Brucella antigen and blood serum can be established. Therefore, the application prospect of the monoclonal antibody is wide.

Description

technical field [0001] The invention relates to a hybridoma cell strain 17C8 obtained by immunizing mice with the whole brucella as an antigen and a monoclonal antibody produced therefrom. Background technique [0002] Brucellosis is a zoonotic disease caused by Brucella bacteria. Brucella can cause abortion, infertility, orchitis and arthritis in mammals, and it is also used as a biological warfare agent because it easily forms aerosols and is highly infectious. [0003] Traditional Brucella detection adopts serological methods, mainly including rosette test, test tube agglutination test (SAT), tiger red plate agglutination test (RBPT), complement fixation test (CFT), antihuman globulin test (COOMB'S) , and ELISA methods that can be standardized, etc. The downside of serological methods is that it is less sensitive and prone to cross-reactivity with Vibrio cholerae, Yersinia enterocolitica, EHEC, Salmonella, Shigella flexneri, etc. Therefore, the preparation of monoclona...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/577
Inventor 汪舟佳陈泽良王玉飞于爽杜昕颖苑锡铜袁静黄留玉
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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