Monoclonal antibody for identifying alternaria tenuissima, and hybridoma cell strain AtD2 of monoclonal antibody

A hybridoma cell line and monoclonal antibody technology, applied in the field of animals, can solve problems such as agricultural production losses, and achieve the effect of strong specificity and good development and application prospects

Active Publication Date: 2020-02-18
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alternaria tenugarii is also the causative bacterium of black spot disease

Method used

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  • Monoclonal antibody for identifying alternaria tenuissima, and hybridoma cell strain AtD2 of monoclonal antibody
  • Monoclonal antibody for identifying alternaria tenuissima, and hybridoma cell strain AtD2 of monoclonal antibody
  • Monoclonal antibody for identifying alternaria tenuissima, and hybridoma cell strain AtD2 of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Preparation of hybridoma cell lines

[0019] (1) Pick a single colony of Alternaria tenopolaris and inoculate them in potato dextrose liquid medium, shake and culture at 25°C for 4-5 days, collect spores and hyphae in a 50mL centrifuge tube, and centrifuge at 6000r / min for 20min , after washing twice with PBS, ultrasonic crushing (power 200W, crushing 2s, intermittent 2s), the crushed solution was centrifuged at 6000r / min for 20min, the supernatant was collected, and the supernatant protein content was measured by the Coomassie brilliant blue method, and the protein concentration was adjusted. 1000μg / mL, as the initial antigen for immunization antigen and later detection, the antigen solution was aliquoted in a small amount and stored in a freezer at -80°C. Take a small amount and store at -20°C before use.

[0020] (2) Three healthy BaL b / c mice aged 10-12 weeks were selected and injected intraperitoneally with 200 μL of antigen emulsified with an equal ...

Embodiment 2

[0023] Embodiment 2: the production of monoclonal antibody

[0024] Take BaL b / c mice about 8 weeks old, inject 0.3mL pristane intraperitoneally, and inject 5-10×10 5 7-10 days after injection, the peritoneal cavity of the mouse was obviously swollen. The ascites was collected, centrifuged at 2000r / min for 3min, and the supernatant was collected, which was the ascitic monoclonal antibody. Monoclonal antibodies were purified by Protein A column chromatography and stored at -80°C. The monoclonal antibody prepared from the AtD2 cell line is a monoclonal antibody that can specifically recognize Alternaria tenugarius.

Embodiment 3

[0025] Example 3: Titer detection experiment of monoclonal antibody

[0026] Antibody titer was determined by indirect ELISA method. Dilute 1000 μg / mL Alternaria tenugarii antigen 1000 times with coating solution and coat the whole microplate plate (1 μg / mL), overnight at 4°C, let it adsorb to the wells of the polystyrene plate, wash with PBST After three times, it was blocked with skim milk for 60 min. Monoclonal antibody AtD was diluted 2 times and added to the coated wells, 100 μL was added to each well, 37°C, 1 h, washed three times with PBST, and horseradish peroxidase-labeled rabbit anti-mouse (Sigma Company) diluted 5000 times according to the instructions was added to 100 μL wells, 37 ℃1h, after washing with PBST, add OPD substrate chromogenic solution to develop color, use 50μL 2M H 2 SO 4 After terminating the reaction, read the OD with a microplate reader 490nm , To determine the titer of monoclonal antibody ascites with the ratio of negative to positive greater...

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Abstract

The invention discloses a monoclonal antibody for identifying alternaria tenuissima, and a hybridoma cell strain of the monoclonal antibody. The monoclonal antibody for identifying alternaria tenuissima is secreted and generated by a hybridoma cell strain a the preservation number of CCTCC No:C2019228. The hybridoma cell strain is named as a hybridoma cell strain AtD2, is preserved in China Centerfor Type Culture Collection (CCTCC) on Oct.17,2019, and the preservation number of the hybridoma cell strain is CCTCC No:C2019228. The monoclonal antibody is used for identification and dynamic monitoring of animal and plant diseases caused by infection of alternaria tenuissima and biological research of alternaria tenuissima, and a large amount of the monoclonal antibodies can be obtained by carrying out intraperitoneal injection on a BaLb/c mouse. Compared with polyclonal antibodies, the monoclonal antibody has the advantages of high purity, strong specificity, good repeatability and the like.

Description

technical field [0001] The invention belongs to the field of animals, and in particular relates to a monoclonal antibody for recognizing Alternaria tengella and its hybridoma cell line AtD2. Background technique [0002] Alternaria tenuissima is one of the pathogenic fungi of Fritillaria tenuissima. The fungus survives the winter in the soil with mycelia and remains in the soil with the sick tissue, and infects the Fritillaria again in the second year. Alternaria tenugarii is also the causative bacterium of black spot disease of many crops, which causes serious losses to agricultural production every year. However, there are many kinds of Alternaria fungi, and the colonies, hyphae, and spores of related species are similar in shape, so it is difficult to distinguish them based on the above characteristics. The monoclonal antibody (mAb) combined with the enzyme-linked immunosorbent assay (ELISA) disclosed by the present invention against Alternaria tengella has the characte...

Claims

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Application Information

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IPC IPC(8): C07K16/14C12N5/20C12R1/91
CPCC07K16/14
Inventor 赵伟春徐云飞李吉二王海林
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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