Yersinia enterocolitica monoclonal antibody hybridoma cell strain and application thereof
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A hybridoma cell line, enterocolitis technology, applied in anti-bacterial immunoglobulin, biochemical equipment and methods, instruments, etc., to achieve good specificity
Inactive Publication Date: 2021-06-29
JIANGNAN UNIV
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Embodiment 1
[0021] The preparation of embodiment 1 hybridoma cell line JSL 3H3
[0022] (1) Preparation of complete antigen: Yersinia enterocolitica was inserted into 100mL LB broth medium, stirred overnight at 180rpm at 28°C, and then the overnight culture of Yersinia enterocolitica was incubated at 100 Heat inactivation in a water bath at ℃ for 15min, centrifuge at 5000×g for 20min at 4℃, harvest bacteria, wash with sterile phosphate buffer saline, resuspend in 5mL PBS, and store at -20℃.
[0023] (2) Animal immunization: healthy BALB / c mice aged 6-8 weeks were selected for immunization. After the complete antigen of Yersinia enterocolitica was mixed and emulsified with an equal volume of Freund's adjuvant, BALB / c mice were immunized by subcutaneous injection in the back. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second booster immunization was 28 days, and the interval ...
Embodiment 2
[0032] Example 2 Application of Yersinia enterocolitica monoclonal antibody
[0033] The monoclonal antibody prepared by the hybridoma cell line JSL 3H3 through the peritoneal fluid of mice was applied to the detection of Yersinia enterocolitica, and the specific steps of ic-ELISA to detect the antibody titer were as follows:
[0034] (1) Coating: Yersinia enterocolitica was used 0.05M pH9.6 carbonate buffer solution from 10 8 CFU / mL start doubling dilution, 100μL / well, react at 37℃ for 2h;
[0035] (2) Washing: Pour off the solution in the plate, and wash with washing liquid 3 times, each time for 3 minutes;
[0036] (3) Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 hours. After washing, dry it for later use;
[0037] (4) Adding samples: Dilute the antibody starting from 1:1000, and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 minutes; after fully washing, add 1:3000 diluted HRP- Goat anti-mous...
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Abstract
The invention discloses a yersinia enterocolitica monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection. The yersinia enterocolitica monoclonal antibody hybridoma cell strain JSL3H3 is classified and named as a monoclonal cell strain, the preservation date is September 27, 2020, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.20786. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity for yersinia enterocolitica. The yersinia enterocolitica complete antigen is synthesized, the complete antigen and an equal amount of a freund's adjuvant are mixed and emulsified completely, a BALB / c mouse is immunized through back subcutaneous injection, and the hybridoma cell strain JSL 3H3 is obtained through screening of an indirect competitive enzyme-linked immunosorbent assay and three times of subcloning. The monoclonal antibody secreted by the cell strain has good specificity for the yersinia enterocolitica, and the results of the invention can be used for specific detection of yersinia enterocolitica in food, and has practical application value.
Description
technical field [0001] The invention relates to a Yersinia enterocolitica monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immunodetection. Background technique [0002] The genus Yersinia belongs to the Enterobacteriaceae family and consists of three human-pathogenic bacteria: Yersinia pestis (the pathogen of plague), Yersinia pseudotuberculosis and Yersinia responsible for enteric Yersiniasis Yersinia enterocolitica. Yersinia enterica is a foodborne illness caused by consumption of contaminated food or water and can be transmitted from person to person through the fecal-oral route. Yersinia enterica is usually characterized by a self-limited acute infection that begins in the gut and is usually limited to the ileocecal junction. Diarrhea is the main symptom of Yersinia enterocolitica infection. In addition, Yersinia enterocolitica can also cause different clinical symptoms, such as rash, conjunctivitis, acute orga...
Claims
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