Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN)

A technology for zearalenone and a detection method, which is applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problems of increasing the discharge of toxic and harmful substances and harming operators.

Active Publication Date: 2012-12-26
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ZEN is a small molecule toxin. The immunological method currently used for the quantitative detection of ZEN residues requires the use of ZEN standards. The use of ZEN standards not only brings harm to ope

Method used

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  • Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN)
  • Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN)
  • Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of Zearalenone Monoclonal Antibody

[0073] 1. Preparation of immune antigen ZEN-OVA and detection antigen ZEN-BSA

[0074] The active ester method was used to couple ZEN to OVA to prepare the immune antigen (ZEN-OVA), and to couple ZEN to BSA to prepare the detection antigen (ZEN-BSA).

[0075] ZEN is derived from ZEN-CMO

[0076] (1) Weigh 6.5mg of ZEN, add 1ml of pyridine, then add 13mg of O-carboxymethylhydroxylamine, stir at room temperature for 24 hours, and finally vacuum dry.

[0077] (2) Dissolve the dissolved residue in 4m distilled water, and adjust the pH to 8.0 with 1M NaOH.

[0078] (3) Extract twice with 4ml of benzene to remove unreacted ZEN, then adjust the pH to 3.0 with 1M HCl.

[0079] (4) Extract 4 times with 8ml ethyl acetate, collect the extract, and vacuum dry, the residue is zearalenone carboxymethyl oxime (ZEN-CMO)

[0080] Synthesis of ZEN-BSA:

[0081] (1) Preparation of activated ester: Weigh 3.0mg ZEN-CMO, dissolv...

Embodiment 2

[0092] Example 2 Preparation of zearalenone anti-idiotypic monoclonal antibody

[0093] 1. Using the monoclonal antibody secreted by the hybridoma cell 1G4G10D3, which stably secretes highly sensitive anti-ZEN monoclonal antibody, as the immunogen, mice were immunized, and the cells were fused to prepare the zearalenone anti-idiotypic monoclonal antibody.

[0094] (1) Preparation of immunogen 1G4G10D3-KLH conjugate

[0095] ① Add 25.6 mg EDC to 4 mg / mL IgG solution (PBS, 500 μL), and stir at room temperature for 30 min;

[0096] ②Add 2 mg / mL KLH solution (PBS, 1 mL) to the above solution, stir and react at 4°C for 12 h~16 h;

[0097]③ The final reaction solution was dialyzed against 0.01 M PBS for two days to obtain the immunogen mAb-KLH conjugate (ie 1G4G10D3-KLH conjugate), which was aliquoted and frozen at -20°C for later use.

[0098] (2) Preparation and identification of the Fab antibody fragment of monoclonal antibody 1G4G10D3

[0099] ① Preliminary purification of ZE...

Embodiment 3

[0109] Example 3 Establishment of Zearalenone Competitive ELISA Nontoxic Detection Method

[0110] 1. Preparation of pre-coated strips:

[0111] (1) Coating: Dilute the detection antigen ZEN-BSA to 1 μg / mL with coating buffer, namely 0.05M sodium carbonate buffer solution at pH 9.6, add 100 μL per well into a polystyrene microwell plate, and keep at 37°C Incubate for 3 hours;

[0112] (2) Washing: After pouring out the coating buffer in the wells of the microplate, add 200 μl of washing solution into the wells with a washing bottle or a multi-channel pipette; pour out the liquid in the wells again after 3 to 5 minutes to completely remove liquid in the well; wash 3 to 5 times; the washing solution is PBS-T, which contains 0.05% Tween-20 0.01M phosphate buffered saline (KH 2 PO 4 0.2g, Na 2 PO 4 12H 2 O 2.9g, NaCl 8g, dilute to 1000ml, pH7.4, add 0.5ml Tween-20);

[0113] (3) Blocking: Add 150-200 μL of 0.01M PBS (phosphate buffered saline, pH 7.4) containing 0.5%-3% BS...

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Abstract

The invention discloses a competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone. The method disclosed by the invention is a competitive ELISA method built by ZEN-BSA (zearalenone-bovine serum albumin), a ZEN monoclonal antibody and a beta-anti-idiotype monoclonal antibody of ZEN. A ZEN standard substance is replaced by the beta-anti-idiotype monoclonal antibody of ZEN, so as to avoid the damage of toxin to an operator and the possibility of scattering toxin to the environment, and non-toxic detection is achieved. According to the method, the ZEN standard substance is not needed, so that the cost can be greatly reduced; and zearalenone polluted by foods, grains, feed and the like can be rapidly and sensitively detected at a large scale, therefore, the method has a good market prospect.

Description

technical field [0001] The invention relates to the field of compound detection, in particular to a nontoxic competitive ELISA detection method for zearalenone. Background technique [0002] Zearalenone (Zenralenone, ZEN) is an estrogen-like mycotoxin produced by a variety of Fusarium fungi. Many links such as storage and use may be polluted by ZEN. ZEN has strong reproductive toxicity and teratogenic effects, which can cause hyperestrogenism in animals, lead to infertility or abortion, and have a great impact on pigs, poultry, and ruminants, and bring great economic losses to animal husbandry; ZEN It also has cytotoxicity, immunotoxicity, hepatotoxicity, potential carcinogenicity, etc., and ZEN is stable to high temperature, not easy to remove, and seriously endangers human health. [0003] In view of the strong toxic effect of zearalenone on humans and other animals, JECFA announced in 2000 that the temporary maximum intake of ZEN for humans is 40 μg / kg body weight. As ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/545C07D313/00
Inventor 王宏宋其芳
Owner JINAN UNIVERSITY
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