Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin

A technology of aflatoxin and detection method, applied in the field of aflatoxin detection, can solve the problems of operator and environmental hazards, high price, tight supply, etc.

Inactive 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

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Problems solved by technology

Moreover, the use of these highly toxic and strong carcinogens will bring great harm to operators and the environment, and because of high price and tight supply, it often affects the detection process

Method used

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  • Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin
  • Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin
  • Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The preparation of embodiment 1 standard substance universal substitute

[0047] 1. Hybridoma cell line 3G1, which was deposited in the China Center for Type Culture Collection (CCTCC) on July 13, 2010. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO. C201014. Its screening method, preparation of anti-aflatoxin B1 monoclonal antibody and its subtype characteristics are as follows:

[0048] (1) Screening of hybridoma cell line 3G1

[0049] ①Antigen synthesis and animal immunization

[0050] 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 or...

Embodiment 2-4

[0088] In following embodiment 2-4:

[0089] The coating buffer is: Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, add pure water to make up to 1 L.

[0090] The OVA is ovalbumin.

[0091] The PBST solution is: KH 2 PO 4 0.2 g, KCl 0.2 g, Na 2 HPO 4 12H 2 O 2.9 g, NaCl 8.0 g, 0.5 mL Tween-20, add pure water to make up to 1 L.

[0092] The chromogenic solution is 9.5 mL of substrate buffer + 0.5 mL of substrate use solution + 32 μL of 3% hydrogen peroxide

[0093] composition.

[0094] The substrate buffer is: Na 2 HPO 4 12H 2 O 1.841 g, C 6 h 7 o 8 ·H 2 O 0.933 g was dissolved in 100 mL ultrapure water.

[0095] The substrate used solution is: 2 mg TMB dissolved in 100 mL absolute ethanol.

Embodiment 2

[0096] Example 2 Determination of Aflatoxin B1 in Peanut Samples

[0097] (1) Establishment of the conversion equation for the corresponding relationship between the concentration of the general substitute of the standard substance and the concentration of the standard substance of aflatoxin B1

[0098] ① Aflatoxin B1 Standard ELISA Standard Analysis Curve Equation

[0099] First dilute aflatoxin B1 complete antigen (AFB 1 -OVA, 2 μg / mL), added to the microtiter plate, 100 μL per well, 4 °C overnight. After washing the plate three times with PBST solution, 200 μL of 1.5% OVA aqueous solution was added to each well as a blocking solution, and reacted at 37 °C for 1 h. Wash the plate three times with PBST, add 50 μL anti-aflatoxin B1 monoclonal antibody solution to each well, and then add aflatoxin B1 standard (2.4 to 600 pg / mL). After reacting at 37 °C for 1 h, the plate was washed three times with PBST, and 100 μL of commercial goat anti-mouse enzyme-labeled secondary antib...

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Abstract

The present invention relates to a standard substance universal alternate for aflatoxin detection by using ELISA, a preparation method thereof, and an ELISA detection method for aflatoxin. The standard substance universal alternate is characterized in that: the standard substance universal alternate is a rabbit anti-mouse antibody capable of recognizing various mouse anti-aflatoxin monoclonal antibodies. The preparation method comprises: adopting a mouse anti-aflatoxin B1 monoclonal antibody, a mouse anti-aflatoxin M1 monoclonal antibody, and a mouse anti-aflatoxin G1 monoclonal antibody as a mixed antigen; adopting the mixed antigen to immunize New Zealand long ear white rabbits; sampling blood from carotid arteries; and carrying out affinity purification on serums to obtain the standard substance universal alternate. The standard substance universal alternate of the present invention is non-toxic and harmless, and can respectively replace various corresponding aflatoxin standard substances in ELISA detection methods for aflatoxin to quantitatively detect various corresponding aflatoxin concentrations in samples.

Description

technical field [0001] The invention belongs to the field of aflatoxin detection, and in particular relates to a general substitute for a standard product used for ELISA detection of aflatoxin, a preparation method thereof and an ELISA detection method of aflatoxin. Background technique [0002] Aflatoxins are mainly produced by Aspergillus flavus ( Aspergillus flavs ), Aspergillus parasitica ( Aspergillus parasiticus ) and Aspergillus fruticosa ( Aspergillus nonius ) produced a group of highly toxic and carcinogenic secondary metabolites, mainly aflatoxin B1 (Aflatoxin B1 1 , AFB 1 ), aflatoxin M1 (Aflatoxin M 1 , AFM 1 ), aflatoxin G1 (Aflatoxin G 1 , AFG 1 ). It widely exists in various agricultural products, food and feed, seriously pollutes peanuts, rice, corn, wheat and other grain and oil products, and greatly threatens people's health and life safety. Due to the increasingly serious potential threat of aflatoxin to humans, more than 100 countries in the wo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C07K16/06G01N33/53
Inventor 李培武管笛张奇张文丁小霞
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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