Method for detection of shigella boydii and monoclonal antibodies

A technology for salmonella and hog cholera, applied in the field of microbial detection, can solve problems such as inability to adapt to samples, screening, and long detection cycle

Active Publication Date: 2014-05-28
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of Salmonella choleraesuis mainly relies on the biochemical identification stipulated in the national standard. The disadvantage is that the operation is cumbersome, the detection cycle is long, and it cannot adapt to the screening of a large number of samples.
Immunological detection is the fastest

Method used

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  • Method for detection of shigella boydii and monoclonal antibodies
  • Method for detection of shigella boydii and monoclonal antibodies
  • Method for detection of shigella boydii and monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Preparation of monoclonal antibodies Mab05-D10 and Mab05-F10 against Salmonella choleraesuis

[0077] 1. Preparation of immunogen and positive standard

[0078] Salmonella choleraesuis (CICC21493) was inoculated on a sorbitol Macconkey (SMAC) plate, cultured at 37°C for 24 hours, and a single colony was picked out in buffered peptone water (BPW), cultured with shaking at 37°C, 150r / min for 17 hours, counted, and added 0.3% The formaldehyde solution is inactivated for 1 day at room temperature. Adjust the concentration of Salmonella cholerae suis (CICC21493) to 5×10 with normal saline 9 cfu / ml as immunogen; adjust the concentration to 10 with saline 8 cfu / ml Salmonella cholerae suis was used as a positive control standard, and buffered peptone water (BPW) was used as a negative control standard.

[0079] 2. Preparation of monoclonal antibodies

[0080] 1) Experimental animals: Choose 3 female Balb / c mice, 8 weeks old, weighing about 20g, as experimental animals.

[00...

Embodiment 2

[0105] Example 2. Characterization of monoclonal antibodies Mab05-D10 and Mab05-F10

[0106] 1. Identification of monoclonal antibody subclasses

[0107] 1. Antigen coating: Use 0.01M PBS to coat goat anti-mouse secondary antibody IgG+A+M, 50μl per well, coat overnight at 4°C, discard the liquid in the well the next day, and wash the plate 3 times.

[0108] 2. Blocking: Add 200μl of 1% BSA to each well and block overnight at 4°C. Pat the board dry the next day without washing it.

[0109] 3. Add the supernatant of monoclonal antibody hybridoma cells, 8 microwells per sample, 50μl per well. Incubate at 37°C for 1h.

[0110] 4. After washing the plate 4 times, add specifically bound rabbit anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, and incubate at 37°C for 1 hour.

[0111] 5. After washing the plate 4 times, add diluted horseradish peroxidase labeled anti-rabbit secondary antibody IgG (H+L) to each well, incubate at 37°C for 30 minutes.

[0112] After washing the plate 4 times, ...

Embodiment 3

[0126] Example 3. Composition, preparation and application of an enzyme-linked immunoassay kit for detecting Salmonella cholerae suis

[0127] 1. The enzyme-linked immunoassay kit consists of the following materials:

[0128] (1) Pre-coated antibody-enzyme-labeled plate: diluted with 0.02M acetate buffer (pH 2.0) solution, anti-S. cholerae monoclonal antibody Mab05-F10 coated 96-well ELISA plate, 100μl per well. Incubate overnight at 4°C, block and wash according to conventional ELISA methods.

[0129] (2) Salmonella choleraesuis positive control standard and negative control standard.

[0130] (3) Horseradish peroxidase-labeled monoclonal antibody Mab05-D10 against Salmonella choleraesuis.

[0131] (4) Enzyme-labeled antibody diluent: 0.01M PBS, pH 7.6.

[0132] (5) 10× concentrated lotion: 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% sodium azide, pH 7.4, just dilute the concentrated lotion 10 times before use.

[0133] (6) Chromogenic liquid A liquid, chromogenic liquid B l...

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Abstract

The invention belongs to the field of microbial detection. Specifically, the invention relates to a method for detection of shigella boydii, and two strains of anti-shigella boydii monoclonal antibodies used in the method and produced in a same-time monoclonal antibody preparation process. The monoclonal antibodies can be specifically combined with shigella boydii, and are produced from a mouse hybridoma cell line Mab05-D10 with CGMCC No.7710 or Mab05-F10 with CGMCC No.7712. The invention also provides a use of the anti-shigella boydii monoclonal antibodies and hybridoma cells for producing the antibodies.

Description

Technical field [0001] The invention belongs to the field of microorganism detection. Specifically, the present invention relates to a method for detecting Salmonella cholerae suis, two strains of anti-Salmonella cholerae suis monoclonal antibodies used in the method and their uses, and hybridoma cells producing the monoclonal antibodies. Background technique [0002] Salmonella choleraesuis (Shigella boydii) belongs to Shigella C group and is an important food-borne pathogen. It can enter the human body through contaminated meat, eggs, fish and other animal products and melon seeds. , Cause fever, abdominal pain, tenesmus, and sometimes manifested as toxic dysentery as symptoms of systemic poisoning, and the treatment is not completely turned into chronic. [0003] At present, the detection of Salmonella choleraesuis mainly relies on the biochemical identification specified by the national standard. Its disadvantages are cumbersome operation and long detection period, which canno...

Claims

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Application Information

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IPC IPC(8): G01N33/577C07K16/12C12N5/20
CPCC07K16/1235G01N33/56916G01N2333/255
Inventor 刘箐邱实
Owner UNIV OF SHANGHAI FOR SCI & TECH
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