Method and kit for rapidly detecting AFB1 by direct competitive ELISA (enzyme-linked immuno sorbent assay) method

A direct and rapid technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of mycotoxins contamination, contamination, low precision of ELISA methods, etc., and achieve the effect of high sensitivity and simple operation.

Pending Publication Date: 2021-07-16
TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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Problems solved by technology

For example, Jiang Jiayi et al. (2020) used ELISA to detect AFB in traditional Chinese medicine 1 , the linear range is 0.05~0.58μg / kg, and the detection limit is 1.69μg / kg, which is in good agreement with the results of UHPLC-MS / MS; the mycotoxin contamination is extensive, Wang Guoqiang et al. (2019) found in the detection of feed and raw materials from different provinces All samples were contaminated, and samples contaminated by three or more toxins were as high as 76.55%; AFB was detected in cassava in Cameroon, Tanzania, Nigeria, etc. 1 reports; although there are AFBs at home and abroad 1 Many rapid detection products have appeared, but the precision of the current ELISA method for aflatoxin B1 is low, and there is no method for analyzing samples of food and its products such as cassava flour

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  • Method and kit for rapidly detecting AFB1 by direct competitive ELISA (enzyme-linked immuno sorbent assay) method
  • Method and kit for rapidly detecting AFB1 by direct competitive ELISA (enzyme-linked immuno sorbent assay) method
  • Method and kit for rapidly detecting AFB1 by direct competitive ELISA (enzyme-linked immuno sorbent assay) method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: a kind of method for the rapid detection of AFB1 of direct competition ELISA method, it comprises the following steps:

[0030]S1. Sample processing: Dry and pulverize the samples of cassava, corn, soybeans and peanuts, and accurately weigh the samples after sieving. Each gram of samples is extracted with 5ml of 70% methanol aqueous solution and 10% of the sample mass with sodium chloride. For cassava, take 2-fold dilution of the supernatant and test it. For corn, soybean and peanut, take 1 mL of the supernatant and add 0.8 mL of n-hexane. After shaking and centrifuging, take the supernatant of the lower layer for 2-fold dilution and test;

[0031] S2, establish standard curve: select 0.05ng / ml inhibition rate more than 50%, OD 450 The dilution factor of AFB1 antibody and AFB1-HRP enzyme-labeled antigen whose value is closest to 2.0 is used as the titer, and the concentration of the standard product is diluted to perform competition ELISA. The logarithm va...

Embodiment 2

[0036] Example 2: Aflatoxin B 1 Preparation and Identification of Antigen and Antibody

[0037] 2.1 Preparation and identification of aflatoxin B1 antigen

[0038] Conjugate preparation: refer to the methods of Deng Juan (2016) and Sun Qing (2017) to synthesize aflatoxin B 1 Oximide (AFB 1 .O), identified correctly by mass spectrometry scanning. Then use the carbodiimide method (DCC method) to AFB 1 .O was coupled with the carrier proteins BSA, OVA and HRP respectively, and then dialyzed in PBS with a pH value of 7.2 for 48 hours, and stored in -20°C.

[0039] Conjugate identification: take the coupling product AFB 1 -BSA, AFB 1 -OVA, AFB 1 -HRP and negative controls BSA, OVA, HRP, and purchased AFB 1 -BSA antigen was coated on the microtiter plate as an antigen, and at the same time coated with PBS as a blank control, with the purchased AFB 1 The antibody was used as the primary antibody, and the HRP-labeled goat anti-rabbit IgG was used as the secondary antibody for...

Embodiment 3

[0056] Embodiment 3: the establishment of competition ELISA detection method

[0057] 3.1 Establishment of aflatoxin B1 standard curve

[0058] Use competitive ELISA checkerboard square matrix to determine titer, select OD 450 The dilution multiple of the antigen, antibody, and enzyme-labeled antigen closest to 2.0 is used as the titer, and the concentration of the standard is diluted to perform competitive ELISA. The logarithmic value of the standard concentration is used as the abscissa, and the logit value of the absorbance value is used as the ordinate to establish ELISA standard curve to obtain linear regression equation and correlation coefficient.

[0059] like image 3 As shown, both the establishment of indirect competition ELISA and direct competition ELISA decreased significantly with the longer reaction time. The direct competition method is more sensitive, the IC50 is lower, more stable, and the sample addition method is simpler, so the direct competition ELISA...

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Abstract

The invention discloses a method for rapidly detecting AFB1 (aflatoxin B1) by a direct competitive ELISA (enzyme-linked immunosorbent assay) method and a kit thereof, and belongs to the field of aflatoxin B1 detection. According to the present invention, the antigen and the monoclonal antibody are successfully prepared, the direct competition ELISA method is established, and the method can detect the AFB1 in cassava flour and cassava products such as cassava biscuits, cassava cakes, cassava vermicelli, cassava noodles and other samples, and can also detect the AFB1 in other grain samples. The method has the characteristics of high sensitivity, simplicity in operation, rapidness, accuracy, capability of detecting samples in batches and the like, and is easily mastered and applied by enterprises and basic-level detection personnel. The detection method provides a rapid detection technology for screening AFB1 pollution in food, and provides theoretical basis and technical support for food processing and utilization and industrial popularization.

Description

technical field [0001] The invention relates to the field of detection of aflatoxin B1, in particular to a method for rapid detection of AFB1 by a direct competition ELISA method and a kit thereof. Background technique [0002] Aflatoxins (Aflatoxins) is a general term for a class of structural analogues produced by Aspergillus flavus and Aspergillus parasites. It exists widely in various grains, foods, and feeds, and has stable physical and chemical properties. The existing agricultural technology and Food processing technology cannot avoid and eliminate pollution. of which aflatoxin B 1 (Aflatoxin B 1 ,AFB 1 ) toxicity and "three causes" (carcinogenic, teratogenic, mutagenic) all rank first. EU regulation AFB 1 The content shall not be higher than 2μg / kg, and the national standard of our country stipulates that AFB in grain 1 The content shall not exceed 5 μg / kg, and the food for special dietary use shall not exceed 0.5 μg / kg (GB 2761-2017). [0003] Cassava is an i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543
CPCG01N33/543G01N33/577G01N2333/38
Inventor 余厚美张振文李其铸王琴飞林立铭徐缓
Owner TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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