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Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same

A hybridoma cell line and aflatoxin technology, which is applied to the methods using virus/cell lines, methods based on microorganisms, microorganisms, etc., can solve the problems of high degree of purification, cumbersome operation, and time-consuming detection process, and achieve specificity Good performance and high sensitivity

Active Publication Date: 2012-10-24
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • 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 aflatoxin detection. This method does not require special equipment and can be carried out in general laboratories, but it has a large consumption of reagents, cumbersome operations, easily disturbed results, and poor accuracy. , cannot be accurately quantified, and it is not suitable for on-site rapid detection.
Precision instrument analysis methods include fluorescence spectrophotometry and high performance liquid chromatography, etc. These methods have high sensitivity and accurate detection results, but the required instruments and equipment are expensive, and the sample pretreatment process is cumbersome, requiring a high degree of purification of aflatoxin samples. The detection process is time-consuming and has high requirements on the experimental environment, making it difficult to achieve rapid detection

Method used

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  • Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same
  • Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same
  • Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: Screening of hybridoma cell line 3G1

[0021] 1. Antigen Synthesis and Animal Immunization

[0022] Purchase commercially available aflatoxin B1 standard substance for complete antigen synthesis, the specific synthesis steps are as follows: dissolve 4 mg AFB1 in 2 mL acetone, add 40 μL 10% H 2 SO 4 , the mixture was stirred at 56 °C for 4 h; after the product was evaporated to dryness, 5 mL of H 2 O, extracted twice with 25 mL chloroform, then with 20 mL H 2 The organic layer was washed with O, and the organic layer was retained; the organic solvent was evaporated to obtain a yellow solid product. Take 1.0 mg product, add 2 mL 0.5% BSA solution (20 mg BSA dissolved in 4 mL PBS (phosphate buffer, pH7.4)) to react at 37 °C for 30 min; add 100 μL 6.5 mM NaBH 4 , react at 4°C for 30 min; add 50 μL of 0.1mol / L HCl to remove excess NaBH 4 . At 4°C, dialyze PBS solution (phosphate buffered saline, pH 7.4) for 3 days to remove AFB1 and AFB 2a , and finally car...

Embodiment 2

[0029] Embodiment 2: Anti-aflatoxin B1 monoclonal antibody hybridoma cell line 3G1 antibody variable region sequence determination

[0030] (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 3G1;

[0031] (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;

[0032] (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

[0034] Example 3: Preparation, purification, subtype and identification of anti-aflatoxin B1 monoclonal antibody

[0035] The anti-aflatoxin B1 monoclonal antibody hybridoma cell line 3G1 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 caprylic 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 present invention relates to a hybridoma cell line 3G1 and an anti-alfatoxin B1 monoclonal antibody produced by the hybridoma cell line 3G1. The hybridoma cell line 3G1 (CCTCCNO.C201014) can be used for preparation of a high titer anti-aflatoxin B1 monoclonal antibody, wherein an enzyme-linked immunosorbent assay (ELISA) method is adopted to determine a titer, and the titer is 6.40*10<6>. The anti-aflatoxin B1 monoclonal antibody of the present invention has characteristics of high sensitivity and good specificity, wherein 50% inhibiting concentration on aflatoxin B1 by the monoclonal antibody is 1.6 ng / mL, cross reaction rate with aflatoxin B2 is 6.4%, and cross reaction rates with aflatoxin G1 and G2 are less than 1%. In addition, the anti-aflatoxin B1 monoclonal antibody of the present invention can be used for determination of aflatoxin B1.

Description

technical field [0001] The invention relates to a hybridoma cell line 3G1 and an anti-aflatoxin B1 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 kinds of aflatoxins have been found so far, among which aflatoxin B1 (AFB1) is the most toxic, its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. There have been many human poisoning incidents caused by AFB1 contamination of the food chain in history. After dairy cows ingest AFB1-contaminated grains, another carcinogen, aflatoxin M1 (AFM1), will be formed after metabolism in the body. AFM1 entering the milk will seriously threaten human health. In addition, AFB1 can also contaminate a variety of raw materials, including fruits, dried fruits, vegetables, condiments, oil crops, to...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/14G01N33/577C12R1/91
CPCC12R1/91G01N33/577C07K16/14G01N2333/38
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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