Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof

A hybridoma cell line, aflatoxin technology, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve problems that have not been reported, and achieve the effect of high application value and high sensitivity

Active Publication Date: 2011-03-30
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

At present, there have been many reports on aflatoxin monoclonal or polyclonal antibodies targeting a singl

Method used

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  • Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof
  • Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof
  • Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Preparation of hybridoma cell line 1C11

[0019] 1. Animal immunization

[0020] Six 6-week-old BALB / c mice were purchased and immunized with commercially available aflatoxin complete antigen AFB1-BSA. For the first immunization, the aflatoxin 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 complete antigen was emulsified with the same amount of aflatoxin in Freund's incomplete adjuvant, and injected intraperitoneally into the mice. 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 of the four immu...

Embodiment 2

[0027] Example 2: Determination of the variable region sequence of the hybridoma cell line 1C11 antibody

[0028] (1) Extraction of total RNA: use the total RNA extraction kit of Tiangen Company and follow the instructions to extract the total RNA that can produce hybridoma cell line 1C11;

[0029] (2) cDNA synthesis: using the total RNA obtained in step 1 as a template, oligo(dT) 15 For primers, follow SuperScript TM -2II Reverse Transcriptase Instructions for reverse transcription to synthesize the first strand of cDNA; primer oligo(dT) 15 Purchased from Invitrogen;

[0030](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 product was separated by 1% aga...

Embodiment 3

[0032] Example 3: Preparation, purification and identification of its subtype and characteristics of anti-aflatoxin universal monoclonal antibody

[0033] The hybridoma cell line 1C11 obtained in Example 2 was injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, the ascites of the mice was collected, and the antibody was purified by octanoic acid-ammonium sulfate method. The specific operation was as follows: Filter mouse ascites with double-layer filter paper, centrifuge at 12,000 r / min for 15 minutes at 4°C, absorb the supernatant, mix the obtained ascites supernatant with 4 times the volume of acetate buffer, and slowly add n-octanoic acid under stirring, the required amount per milliliter of ascites The volume of n-octanoic acid is 33 μL, mixed at room temperature for 30 min, left standing at 4 °C for 2 h, then centrifuged at 12000 r / min for 30 min at 4 °C, discarded the precipitate, filtered the obtained supernatant with double-layer filter paper...

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Abstract

The invention provides a hybridoma cell line 1C11 and an anti-aflatoxin general monoclonal antibody secreted by the same as well as the applications thereof. The hybridoma cell line 1C11 can be used for preparing a high-titer aflatoxin antibody, and a mouse hydroperitoneum antibody is measured to reach 5.12*106 by using an ELISA (Enzyme-Linked Immunosorbent Assay). The anti-aflatoxin general monoclonal antibody has high sensitivity, respectively reaches the IC50 (50% inhibiting concentration) of aflatoxin B1, B2, G1 and G2 to be 1.2, 1.3, 2.2 and 18.0 pg/mL, is the antibody with highest sensitivity among currently reported four aflatoxin antibodies, is used for measuring the total aflatoxin amounts, i.e. the total amounts of the aflatoxin B1, B2, G1 and G2 and has great practical application values.

Description

technical field [0001] The invention relates to a hybridoma cell line 1C11, a universal anti-aflatoxin monoclonal antibody produced by the hybridoma cell line and 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, the most important of which are aflatoxins B1, B2, G1 and G2. [0003] After aflatoxin contaminates food and feed, it directly or indirectly enters the human food chain, threatening human health and life safety, and the degree of harm is directly proportional to the intake of aflatoxin. Aflatoxins are widely present in rice, corn, peanuts, sesame, soybeans, rapeseed and other agricultural products and fish and other foods, and often multiple aflatoxins exist at the same time. For this reason, countries around the world have stip...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/14G01N33/577C12R1/91
Inventor 李培武李鑫张道宏张奇张文丁小霞姜俊陈小媚
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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