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Hybridoma cells of shrimp tropomyosin monoclonal antibodies and application thereof

A tropomyosin and monoclonal antibody technology, applied in the field of hybridoma cells, can solve problems such as restricting the development of allergen detection methods, high cost, and requiring specific analytical instruments for detection.

Active Publication Date: 2018-08-03
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many methods for detecting allergens at present, they all have different problems, such as slow detection speed, high cost, and the need for specific analytical instruments, etc., which restrict the development of allergen detection methods in food.

Method used

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  • Hybridoma cells of shrimp tropomyosin monoclonal antibodies and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The monoclonal antibody preparation of embodiment 1 shrimp tropomyosin

[0036] 1. Preparation of shrimp tropomyosin allergen:

[0037] a. Preparation of shrimp protein extract: Weigh 20 g of peeled shrimp, and process it with a tissue masher to obtain shrimp meat paste. Shrimp paste was immersed in petroleum ether (60-90) at a ratio of 1:10 (W / V) to degrease, stirred overnight at 4°C, centrifuged at 6000 r / min for 15 minutes, discarded the supernatant, and placed the precipitate in a fume hood to evaporate the petroleum ether. Skim shrimp. Immediately immerse in 0.05M pH7.4PBS at a ratio of 1:20 (W / V) for overnight extraction, centrifuge the extract at 7000r / min for 20min at 4°C, discard the precipitate, and the supernatant is the shrimp protein extract.

[0038] b. Fractional precipitation of ammonium sulfate: slowly add saturated ammonium sulfate solid into the shrimp protein extract, stir while adding, until the saturation is 30%, let it stand at 4°C for 1h, centr...

Embodiment 2

[0056] The establishment of embodiment 2 double antibody sandwich ELISA:

[0057] 1) Coating: Coat the microtiter plate with the antibody of cell line number FB12-1 at a concentration of 4 μg / mL, 100 μL / well, overnight at 4 °C;

[0058] 2) Washing: Wash the reaction plate three times with PBST, 3 min each time, 200 μL / well, and then dry the reaction plate;

[0059] 3) Blocking: add 200 μL / well blocking solution, and incubate at 37°C for 2 hours;

[0060] 4) washing: same as 2);

[0061] 5) Sample: Shrimp tropomyosin was diluted to 1 μg / mL with PBS, and then three-fold for serial dilution; another PBS blank control was set. Add 100 μL sample to each well and incubate at 37°C for 1 hour;

[0062] 6) washing: same as 2);

[0063] 7) Add enzyme-labeled antibody (7H6-HRP, 2 μg / mL), 100 μL / well, react at 37°C for 1 hour;

[0064] 8) washing: same as 2);

[0065] 9) Color development: add substrate TMB 100 μL / well, and develop color for 12 minutes;

[0066] 10) Termination: Ad...

Embodiment 3

[0069] Taking shrimp samples as an example to detect the allergenic component shrimp tropomyosin

[0070] 1) Coating: Dilute the mouse anti-shrimp tropomyosin monoclonal antibody to the coating concentration with the coating solution, add 100 μL / well to the microtiter plate, and overnight at 4 °C;

[0071] 2) Washing: Discard the liquid in the wells of the microplate, wash the reaction plate three times with PBST, each time for 3 minutes, 200 μL / well, and then dry the reaction plate;

[0072] 3) Blocking: add 200 μL / well blocking solution, and incubate at 37°C for 2 hours;

[0073] 4) washing: same as 2);

[0074] 5) Sample: gradiently dilute shrimp tropomyosin with sample diluent, and dilute the sample to be tested appropriately, 100 μL per well, set positive control and negative control, and incubate at 37°C for 1 hour;

[0075] 6) washing: same as 2);

[0076] 7) Add enzyme-labeled antibody: Dilute the enzyme-labeled mouse anti-shrimp tropomyosin antibody to the working ...

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Abstract

The invention discloses hybridoma cells of shrimp tropomyosin monoclonal antibodies and application thereof, and belongs to the field of food safety immunodetection. Hybridoma cell strains FB12-1 of the shrimp tropomyosin monoclonal antibodies are preserved in China Committee for Culture Collection Center for general microbiology on September 5th, 2017, the preservation number is CGMCCNo.14690, and the preservation address is Institute of Microbiology, the Chinese Academy of Sciences, No.3, Courtyard No.1, West Beichen Road, Chaoyang District, Beijing City. Antibodies 7H6 secreted by the cellstrains FB12-1 are coated with antibodies; HRP (Horse Radish Peroxidase) marked by the antibodies 7H6 are enzyme labelled antibodies (7H6-HRP). By using shrimp tropomyosin as a standard product, a double-antibody sandwich enzyme-linked immunodetection method for detecting the shrimp tropomyosin is built, and hybridoma cells are applied to detection of food allergens and have practical applicationvalues.

Description

technical field [0001] The invention relates to a hybridoma cell of shrimp tropomyosin monoclonal antibody and an application thereof, belonging to the technical field of food safety immune detection. Background technique [0002] In recent years, food allergy has become a public food safety problem. Food allergy is a common immune disease in humans. It is mainly caused by protein in food and is a type I hypersensitivity reaction mediated by IgE. Its clinical symptoms mainly include edema, dermatitis, asthma, and intestinal syndrome. The detection and quantification of trace allergens in complex food samples has become a top priority. Crustaceans are one of the eight major food allergy-prone foods proposed by the Food and Agriculture Organization of the United Nations. The main allergen of crustaceans is shrimp tropomyosin with a molecular mass of about 36-38kDa. At present, the detection methods for trace allergens in vitro include immunological methods, proteomics method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/18G01N33/68G01N33/577
CPCC07K16/18C12N5/163G01N33/577G01N33/68G01N2333/43508
Inventor 匡华曾露胥传来徐丽广刘丽强宋珊珊吴晓玲胡拥明
Owner JIANGNAN UNIV
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