Hybridoma cell strain 1C8 and anti-aflatoxin G1 monoclonal antibody produced by same

A hybridoma cell line, aflatoxin technology, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of cumbersome treatment process, cumbersome operation, interference of other components, etc., and achieve good specificity , the effect of high sensitivity

Active Publication Date: 2012-10-10
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Among them, thin-layer chromatography is the most commonly used detection method for the detection of aflatoxin earlier. This method does not require special equipment and can be carried out in general laboratories, but the amount of reagents is large, the operation is cumbersome, and other components interfere seriously. , poor accuracy, cannot be accurately quantified, and is more harmful to the experimenters and the surrounding environment, so it is not suitable for on-site rapid detection
Precision instrument analysis mainly includes fluorescence spectrophotometry and high-performance liquid chromatography. These methods have high sensitivity and good accuracy, but they are expensive and require a high degree of purification of aflatoxin samples. The sample pretreatment process is cumbersome and time-consuming. High requirements for the experimental environment, it is difficult to achieve rapid detection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Preparation of hybridoma cell line 1C8

[0017] 1. Antigen Synthesis and Animal Immunization

[0018] Purchase commercially available aflatoxin G1 standard items, and carry out the synthesis of AFG1-BSA complete antigen according to the synthetic method of AFG1-BSA disclosed in Example 1 of the specific embodiment in CN101270146.X;

[0019] Six 6-week-old female BALB / c mice were purchased and immunized with self-synthesized aflatoxin G1 complete antigen AFG1-BSA. For the first immunization, the complete antigen AFG1-BSA was mixed and emulsified with an equal amount of Freund's complete adjuvant, and then injected subcutaneously at multiple points on the back of the mouse. The second immunization was carried out 4 weeks after the first immunization, using Freund's incomplete adjuvant and equal volume of complete antigen AFG1-BSA emulsified, intraperitoneal injection of mice. The interval between the third immunization and the second immunization is 4 week...

Embodiment 2

[0024] Example 2: Determination of the variable region sequence of the anti-aflatoxin G1 monoclonal antibody hybridoma cell line 1C8 antibody

[0025](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 1C8;

[0026] (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;

[0027] (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...

Embodiment 3

[0029] Example 3: Preparation, purification, subtype and identification of anti-aflatoxin G1 monoclonal antibody

[0030] The anti-aflatoxin G1 monoclonal antibody hybridoma cell line 1C8 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 relates to a hybridoma cell strain 1C8 and an anti-aflatoxin G1 monoclonal antibody produced by the same. The hybridoma cell strain 1C8 (CCTCC NO: C201017) provided by the invention can be used for preparing a high titer anti-aflatoxin G1 monoclonal antibody. The titer of an anti-aflatoxin G1 mouse ascites antibody measured by enzyme-linked immunosorbent assay (ELISA) can reach 8.19*10<5>. The anti-aflatoxin G1 monoclonal antibody provided by the invention has high sensitivity and good specificity, has a 50% inhibition concentration IC50 for aflatoxin G1 being 13.92 ng/mL, has no cross reaction with aflatoxin B1, aflatoxin B2, or aflatoxin M1, and can be used for the determination of G1 aflatoxin.

Description

technical field [0001] The invention relates to hybridoma cell line 1C8 and the anti-aflatoxin G1 monoclonal antibody produced therefrom. 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 types of aflatoxins have been discovered so far, mainly including aflatoxin B1 (AFB1), B2 (AFB2), AFG and M1 (AFM1). Among them, AFB1 is the most toxic. Its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. The toxicity of AFG is second. These toxins have strong carcinogenicity. The detection rate in the diet is high. For this reason, countries all over the world have limited the content of AFG in food and so on in the field of food safety. my country is an area with heavy aflatoxin pollution. Therefore, it is of great significance to strengthen the detection of aflatoxin G in agri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/14G01N33/577C12R1/91
Inventor 李培武李鑫张奇丁小霞张文李冉张兆威
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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