Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof

A hybridoma cell line and aflatoxin technology, applied in the fields of application, microbiology, biochemical equipment and methods, can solve the problems of cumbersome operation, cumbersome processing, and long time consumption, and achieve high sensitivity and good specificity

Active Publication Date: 2011-10-19
OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, thin-layer chromatography is the most commonly used detection method for detecting aflatoxins. It does not require special equipment and can be carried out in general laboratories. It is accurate and quantitative, and it is harmful to the experimenters and the surrounding environment, so it is not suitable for rapid on-site detection
Precision instrument analysis methods include fluorescence spectrophotometry and high performance liquid chromatography, which have high sensitivity and good accuracy, but the instruments are expensive, requiring a high degree of purification of aflatoxin samples, and the sample pretreatment process is cumbersome and time-consuming. High, difficult to achieve rapid detection

Method used

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  • Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof
  • Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof
  • Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof

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Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1: the preparation of hybridoma cell line 2C9

[0020] 1. Animal immunization

[0021] Six 6-week-old BALB / c mice were purchased and immunized with commercially available aflatoxin M1 complete antigen AFM1-BSA. For the first immunization, the aflatoxin M1 complete antigen was emulsified with an equal amount of Freund's complete adjuvant, and then injected subcutaneously at multiple points on the back of the neck of the mouse. The second immunization was carried out 4 weeks later, and the mouse was injected intraperitoneally with Freund's incomplete adjuvant and the same amount of aflatoxin M1 complete antigen emulsification. The interval between the third immunization and the second immunization was 4 weeks, and the immunization method was the same. The fourth immunization was carried out 3 weeks after the third immunization, and the immunization method was the same as the second immunization, which was also intraperitoneal injection. The doses for the four...

Embodiment 2

[0026] Embodiment 2: Anti-aflatoxin M1 monoclonal antibody hybridoma cell line 2C9 antibody variable region sequence determination

[0027] (1) Extract total RNA: use the total RNA extraction kit from Tiangen Company and follow the instructions to extract the total RNA that can produce hybridoma cell line 2C9;

[0028] (2) cDNA synthesis: using the total RNA obtained in step 1 as a template, oligo (dT) 15 For primers, follow SuperScript TM -2 II Reverse Transcriptase Instructions for reverse transcription to synthesize the first strand of cDNA; primer oligo (dT) 15 Purchased from Invitrogen;

[0029] (3) Cloning of variable region genes by PCR method: Design primers according to the conserved sites of mouse antibody gene sequences in GENEBANK, and use cDNA as a template to amplify antibody light and heavy chain variable region genes. The PCR program was: 94°C for 30s, 55°C for 1min, 72°C for 1min, 30 cycles of amplification, and finally 72°C for 10min. After the PCR produ...

Embodiment 3

[0031] Example 3: Preparation, purification, subtype and identification of anti-aflatoxin M1 monoclonal antibody

[0032] The anti-aflatoxin M1 monoclonal antibody hybridoma cell line 2C9 obtained in Example 2 was injected into BALB / c mice that had been treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected, and octanoic acid-ammonium sulfate was used to The antibody was purified by the method, and the specific operation was as follows: filter the mouse ascites with double-layer filter paper, centrifuge at 12000r / min for 15min at 4°C, absorb the supernatant, mix the obtained ascites supernatant with 4 times the volume of acetate buffer, and stir slowly Add n-octanoic acid, the volume of n-octanoic acid required per milliliter of ascites is 33 μL, mix at room temperature for 30 minutes, let stand at 4°C for 2 hours, then centrifuge at 12,000 r / min for 30 minutes at 4°C, discard the precipitate, and filter the obtained supernatant with dou...

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Abstract

The invention provides a hybridoma cell strain 2C9, an anti-aflatoxin M1 monoclonal antibody produced by the secretion of the hybridoma cell strain 2C9 and application thereof. The hybridoma cell strain 2C9 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO.C201018. The hybridoma cell strain 2C9 can be used for preparing high-titer anti-aflatoxin M1 monoclonal antibody, and a method of rat ascites antibody ELISA (Enzyma-linked Immunosorbent Assay) is adopted to detect the titer which can reach 4.26*106. The ant-aflatoxin M1 monoclonal antibody has high sensitivity, the 50% of inhibition concentration IC50 aflatoxin M1 caused by the anti-aflatoxin M1 monoclonal antibody is 67pg / ml, and the cross reaction rates between the anti-aflatoxin M1 monoclonal antibody and aflatoxin B1, between the anti-aflatoxin M1 monoclonal antibody and aflatoxin B2, between the anti-aflatoxin M1 monoclonal antibody and aflatoxin G1 and between the anti-aflatoxin M1 monoclonal antibody and aflatoxin G2 are respectively less than 0.1%. The anti-aflatoxin M1 monoclonal antibody can be used for quick detection of aflatoxin M1.

Description

technical field [0001] The invention relates to hybridoma cell line 2C9, the anti-aflatoxin M1 monoclonal antibody produced by it and the application thereof. Background technique [0002] Aflatoxins are mainly secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus, and are natural toxic compounds that can cause various damages to humans and animals. More than 20 kinds of aflatoxins have been found so far, among which aflatoxin B1 is the most toxic, its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of AFB1. After mammals ingest AFB1-contaminated feed, it will be secreted in milk after hydroxylation in the body. Typically, when animals ingest AFB1-contaminated food, the excretion of AFM1 is 1%–3% of the AFB1 intake. A large number of researchers have conducted in-depth studies on the toxicity and carcinogenicity of AFM1, and the results of the research have also prompte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/14G01N33/577C12N15/06
Inventor 李培武管笛李鑫张奇张文丁小霞姜俊陈小媚
Owner OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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