Monoclonal Antibody Recognizing Alternaria and Its Hybridoma Cell Line aaa1

A hybridoma cell line and monoclonal antibody technology, applied in the animal field, can solve problems such as agricultural production losses and achieve strong specific effects

Active Publication Date: 2021-05-18
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alternaria is also the causative bacterium of black spot disease of many crops, causing serious losses to agricultural production every year

Method used

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  • Monoclonal Antibody Recognizing Alternaria and Its Hybridoma Cell Line aaa1
  • Monoclonal Antibody Recognizing Alternaria and Its Hybridoma Cell Line aaa1
  • Monoclonal Antibody Recognizing Alternaria and Its Hybridoma Cell Line aaa1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Preparation of hybridoma cell lines

[0019] (1) Antigen preparation: Pick single colonies of Alternaria 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, 6000r / min Centrifuge for 20 min, wash twice with PBS, ultrasonically crush (power 200W, crush for 2 s, intermittently for 2 s), centrifuge the crushed solution at 6000 r / min for 20 min, collect the supernatant, measure the supernatant protein content with the Coomassie brilliant blue method, adjust The protein concentration is 1000 μg / mL, and it is used as the initial antigen for immunization antigen and later detection. The antigen solution is aliquoted in a small amount and then 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 a...

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 AaA1 cell line is a monoclonal antibody that can specifically recognize Alternaria.

Embodiment 3

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

[0026] Antibody titer was determined by indirect ELISA method. Dilute 1000μg / mL Alternaria antigen 1000 times with the coating solution and coat the whole plate (1μg / mL), overnight at 4°C, make it adsorb to the wells of the polystyrene plate, wash with PBST three times Block with skimmed milk for 60 min. Add monoclonal antibody AaA1-fold dilution to coated wells, add 100 μL to each well, wash at 37°C for 1 h, wash three times with PBST, then add 100 μL of horseradish peroxidase-labeled rabbit anti-mouse (Sigma Company) diluted 5000 times according to the instruction manual, 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 than 2.

[0027] After testing, it was determi...

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Abstract

The invention discloses a monoclonal antibody that recognizes Alternaria and a hybridoma cell line thereof. The monoclonal antibody that recognizes Alternaria is secreted by a hybridoma cell strain with a deposit number of CCTCC No: C2019218 ; The hybridoma cell line is named AaA1, and was deposited in the China Center for Type Culture Collection (CCTCC) on October 17, 2019, and the preservation number is: CCTCC No: C2019218. This monoclonal antibody is used for the identification and dynamic monitoring of animal and plant diseases caused by Alternaria infection and the biological research of Alternaria. It can be obtained by intraperitoneal injection of this cell line into BaL b / c mice Compared with polyclonal antibodies, a large number of monoclonal antibodies have the advantages of high purity, strong specificity, and good repeatability.

Description

technical field [0001] The invention belongs to the field of animals, in particular to a monoclonal antibody for recognizing Alternaria and a hybridoma cell strain thereof. Background technique [0002] Alternaria alternata is one of the pathogenic fungi of fritillaria black spot. 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 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 enzyme-linked immunosorbent assay (ELISA) for Alternaria disclosed by the present invention has the characteristics of high sensitivity, strong specificit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/14C12N5/20
CPCC07K16/14C07K2317/33
Inventor 赵伟春徐云飞祁依佳
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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