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Affinity adsorption material based on anti-aflatoxin nanometer antibody

A technology of aflatoxin and adsorption materials, applied in the field of immunoaffinity adsorption materials, which can solve the problems of reduced activity and intolerance to organic reagents, and achieve the effects of high temperature resistant production, avoiding physical injury, and reducing production costs

Inactive Publication Date: 2016-03-16
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently used aflatoxin affinity purification media generally use polyclonal antibodies or monoclonal antibodies as ligands, which have the problems of being intolerant to organic reagents and rapidly reducing activity

Method used

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  • Affinity adsorption material based on anti-aflatoxin nanometer antibody
  • Affinity adsorption material based on anti-aflatoxin nanometer antibody
  • Affinity adsorption material based on anti-aflatoxin nanometer antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Construction of an immune library of anti-aflatoxin single-domain heavy-chain antibody (single-domain heavy-chain antibody against aflatoxin)

[0021] aflatoxin B 1 Covalently coupled with keyhole limpethemocyanin (KLH) to obtain aflatoxin artificial antigen AFB 1 - KLH, take 300 μg AFB 1 - After KLH was emulsified with complete Freund's adjuvant, the alpaca (Lamapacos) was immunized by subcutaneous multi-point injection. Booster immunization with 150μg AFB 1 -KLH was emulsified with Freund's incomplete adjuvant at intervals of 2 weeks. Blood was collected 7 days after each immunization, and the serum titer was determined by indirect ELISA method. The sample with the highest serum titer was selected to separate lymphocytes and extract RNA.

[0022] The extraction of RNA was carried out according to the instruction manual of RNAiso reagent from TAKARA company. Using RNA as a template and oligodT as a primer, the first strand of cDNA was synthesized according to the i...

Embodiment 2

[0032] Panning and Identification of Anti-Aflatoxin Single Domain Heavy Chain Antibody

[0033] The single-domain heavy-chain antibody against aflatoxin was panned from the anti-aflatoxin single-domain heavy-chain antibody immune library library obtained in Example 1 by using solid-phase affinity panning. will AFB 1 Covalently coupled with ovalbumin (OVA) to obtain the artificial antigen AFB 1 - OVA. Add 100 μL artificial antigen AFB diluted with PBS to each well 1 -OVA, 4°C, coated overnight, the coating concentration of each round of panning was 100, 75, 50 μg / mL; aspirate the coating solution, wash the plate with PBS 3 times, add 300 μL 3% BSA-PBS to each well, 37°C , blocked for 2 hours; the plate was washed 6 times with PBS, and 100 μL of phage antibody library (containing about 2×10 11 CFU), 37°C, incubate for 1.5 h; aspirate unbound phage, wash the plate with PBST (containing 0.5% Tween-20) 5 times (increase 5 times for each round), and then wash the plate with PBS ...

Embodiment 3

[0070] Scale Production of Anti-Aflatoxin Single Domain Heavy Chain Antibody

[0071] Encodes anti-AFB 1 Obtaining the DNA fragment of the single domain heavy chain antibody: 1. Using the restriction endonuclease SfiI / NotI, double-digest the phagemid pHEN-anti-AFB 1 Single domain heavy chain antibody gene, agarose gel electrophoresis to recover anti-AFB 1 Single domain heavy chain antibody gene; 2. direct anti-AFB 1 The single domain heavy chain antibody coding sequence was sent to a biotechnology service company for chemical synthesis; 3. Design specific primers and amplify from a cDNA library derived from alpaca (Lamapacos) by PCR technology.

[0072] anti-AFB 1 The single domain heavy chain antibody gene fragment was cloned into the expression vector pET25, identified by PCR and enzyme digestion, and the anti-AFB was constructed 1 E. coli expression plasmid for single domain heavy chain antibody.

[0073] The expression plasmid was transformed into Escherichia coli BL2...

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Abstract

The invention belongs to the technical field of biology, and relates to an immunoaffinity adsorption material based on a single-domain heavy-chain antibody, in particular to an immunoaffinity adsorption material for aflatoxin. The material comprises a carrier and a ligand carried by the carrier, and is characterized in that the single-domain heavy-chain antibody specially recognizing aflatoxin is used as the ligand and has the amino acid sequence shown in SEQ ID NO.:1and the like. The antibody has the advantages of being resistant to acid, alkali and high temperature, easy to produce and the like, and important practical value is achieved for a low-cost immunological detection method which is used for aflatoxin and can be repeatedly used.

Description

technical field [0001] The invention relates to an immunoaffinity adsorption material based on a single-domain heavy chain antibody (also known as nanobody technology), especially an immunoaffinity adsorption material for aflatoxin. technical background [0002] Aflatoxins are highly toxic secondary metabolites mainly produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. Their chemical structures are similar, and they are difuranocoumarin derivatives, mainly including aflatoxin B 1 (AFB 1 ), B 2 (AFB 2 ), G 1 (AFG 1 ), G 2 (AFG 1 ) and M 1 (AFM 1 )Wait. In naturally contaminated food and feed, AFB 1 Pollution is the most common, and its toxicity and carcinogenicity are also the strongest, and it is classified as a class I carcinogen by the Cancer Research Institute of the World Health Organization. Countries and regions around the world have specified strict aflatoxin limit standards, such as AFB in EU infant food 1 The allowable amount is ≤0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/30B01D15/38G01N21/76
CPCB01D15/3804B01D15/3809B01J20/103G01N1/34G01N33/54313G01N33/54326G01N33/558C07D493/14C07D493/22B01J20/24B01J20/281B01J2220/80B01J2220/52C07G99/00
Inventor 涂追许杨
Owner NANCHANG UNIV
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