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Anti-aflatoxin M1 monoclonal antibody immunoadsorbent, immunoaffinity column and preparation method thereof

A monoclonal antibody and aflatoxin technology, applied in the field of food science, can solve problems such as cytotoxicity, carcinogenicity, economic loss, and impact on the safety of dairy products, achieving high affinity and ensuring safety

Inactive Publication Date: 2017-09-22
QIQIHAR UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, AFM1 cannot be regarded as the detoxification product of AFB1. Many experimental studies have shown that AFM1 is cytotoxic and carcinogenic, and is one of the important harmful substances that affect the safety of dairy products.
[0003] Because aflatoxin M1 (abbreviated as AFM1) is resistant to high temperature and has strong stability, it usually cannot be directly removed by high-temperature sterilization during the processing of dairy products. Therefore, there is currently no direct technical method to remove the contamination of AFM1 in milk. The measures to control AFM1 in milk can only be carried out by adopting strict legal residue limit standards and precise detection technology. Once the standard is exceeded, the emulsion polluted by AFM1 needs to be destroyed, often causing huge economic losses
A typical case is that in 2011, the General Administration of Quality Supervision, Inspection and Quarantine announced that the liquid milk produced by Mengniu Dairy (Meishan) Co., Ltd. and Fujian Changfu Dairy Co., Ltd. contained excessive AFM1. economic loss

Method used

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  • Anti-aflatoxin M1 monoclonal antibody immunoadsorbent, immunoaffinity column and preparation method thereof
  • Anti-aflatoxin M1 monoclonal antibody immunoadsorbent, immunoaffinity column and preparation method thereof
  • Anti-aflatoxin M1 monoclonal antibody immunoadsorbent, immunoaffinity column and preparation method thereof

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Embodiment 1

[0040] An anti-aflatoxin M1 monoclonal antibody, the heavy chain of the anti-aflatoxin M1 monoclonal antibody is IgG2b type, the light chain is λ type, and the first 15 amino acid sequences of the amino terminal of the light chain are GVTQESALTTSPGGT, the The sequence of the first 15 amino acids at the amino terminal of the heavy chain is EVILVESGGGLVKPG, and the molecular weight is 150KD; the anti-aflatoxin M1 monoclonal antibody is compatible with aflatoxin M1, aflatoxin M2, aflatoxin B1, aflatoxin B2, The cross-reactivity rates of G1, aflatoxin G2, deoxynivalenol and BSA were 100%, 4.5%, 21.5%, 1.0%, 16.6%, 1.0%, 0%, 0%, respectively, and the affinity constant was 5.5 ×10-10mol / L.

[0041] The preparation process and identification of the above-mentioned anti-aflatoxin M1 monoclonal antibody are as follows:

[0042] 1. Animal immunization program

[0043] Eight 6-week-old BABL / C mice were selected for immunization. The immunogen was the complete antigen AFM1-BSA. Each imm...

Embodiment 2

[0071] An anti-aflatoxin M1 monoclonal antibody immunoadsorbent, the immunoadsorbent includes a solid phase carrier and an anti-aflatoxin M1 monoclonal antibody coupled to the solid phase carrier, and the solid phase carrier contains epoxy Group activated resin.

[0072] The preparation method of the above-mentioned anti-aflatoxin M1 monoclonal antibody immunosorbent is as follows: specifically comprising the following steps:

[0073] 1) Preparation of polymer microspheres:

[0074] Add the mixture of polyvinylpyrrolidone dissolved in absolute ethanol and deionized water into a three-necked flask, stir to form a homogeneous system, then pass in nitrogen to vent, and raise to the required reaction temperature of 70°C; Azobisisobutyronitrile styrene and glycidyl methacrylate monomer reacted for 24 hours under stirring and nitrogen protection, and then terminated the reaction; the polymer product was centrifugally settled, the clear liquid was discarded, and anhydrous ethanol wa...

Embodiment 3

[0080] An immunoaffinity column loaded with an anti-aflatoxin M1 monoclonal antibody immunosorbent, its preparation method comprises the following specific steps: take the column tube, pad a sieve plate, and apply the coupling anti-aflatoxin prepared in claim 3 The immunosorbent of M1 monoclonal antibody was loaded into the column tube, and equilibration buffer (0.1 mol / L, pH 8.0, Tris-HCl) was added dropwise to stabilize the gel surface and seal the bottom of the mouth.

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Abstract

The invention provides an anti-aflatoxin M1 monoclonal antibody immunoadsorbent, an immunoaffinity column and a preparation method thereof. The anti-AFM1 immunoadsorbent comprises a solid phase carrier and an anti-aflatoxin M1 monoclonal antibody conjugated to the solid phase carrier. The solid phase carrier is an epoxy group-containing activated resin. The anti-aflatoxin M1 monoclonal antibody has an IgG2b type heavy chain and a gamma-type light chain. The front 15 amino acid sequence of the amino group end of the light chain is GVTQESALTTSPGGT. The front 15 amino acid sequence of the amino group end of the heavy chain is EVILVESGGGLVKPG. The molecular weight is 150KD. A coupling ratio of the anti-aflatoxin M1 monoclonal antibody and activated resin is 93.4%. The anti-aflatoxin M1 monoclonal antibody immunoadsorbent has a milk aflatoxin M1 adsorption capacity of 100 microgram per milligram antibody. The immunoadsorbent can remove aflatoxin M1 in fresh milk processing without loss of fresh milk nutrition.

Description

technical field [0001] The invention relates to an anti-aflatoxin M1 monoclonal antibody, an immunosorbent and a preparation method thereof, and also relates to a preparation method of an immunoaffinity column for preparing the immunosorbent, which belongs to the field of food science and technology. Background technique [0002] Aflatoxin M1 (Aflatoxin M1, AFM1) mainly exists in milk, so it was originally called "milk toxin". It was discovered by de Iongh et al. and published in the journal Nature in 1964. With the in-depth research, it is found that AFM1 is aflatoxin B1 (Aflatoxin B1, AFB1) which is formed by hydroxylation under the action of cytochrome P450 series enzymes in vivo. It is generally believed that the hydroxyl metabolite of AFB1 is the detoxification form of toxin . However, AFM1 cannot be regarded as the detoxification product of AFB1. Many experimental studies have shown that AFM1 has cytotoxicity and carcinogenicity, and is one of the important harmful su...

Claims

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Application Information

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IPC IPC(8): C07K16/14B01J20/26B01J20/28B01J20/30B01D15/38
CPCB01D15/3809B01J20/265B01J20/28021B01J20/30C07K16/14
Inventor 张小舟裴世春
Owner QIQIHAR UNIVERSITY
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