Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method

A technology for aflatoxin and ochratoxin, which is applied to measuring devices, instruments, scientific instruments, etc., can solve the problems of determination, inability to perform simultaneous operations, unsatisfactory sensitivity and reagent stability, and achieve convenient use and simple structure. Effect

Inactive Publication Date: 2008-08-13
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection sensitivity and reagent stability of commonly used enzyme-linked immunoassays are not satisfactory, and it is not possible to perform OTA and AFB at the same time 1 determination

Method used

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  • Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method
  • Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of kit and detection of corn samples

[0027] Eu 3+ - Preparation of goat anti-rabbit antibody:

[0028] Take 1-2mL of 5g / L goat anti-rabbit antibody dissolved in 50mmol / L, pH7.0PBS, change the buffer condition through PD-10 column, the eluent is 50mmol / L NaCl containing 0.155mol / L NaCl 2 CO 3 -NaHCO 3 pH 8.5 buffer. The protein peaks were collected and quantified by UV absorption analysis (1.46A 280 -0.74A 260 ), dilute the goat anti-rabbit antibody to 2g / L with the eluent above. Take 500μL diluted goat anti-rabbit antibody and add 0.2mg Eu 3+ -N 2 -[p-isocyanate-benzyl]-diethylenetriaminetetraacetic acid (Eu 3+ -DTTA) vial, 30 ℃ magnetic stirring reaction for 20 hours. The reaction solution was chromatographed on a Sepharose CL-6B column (1×40cm) equilibrated with 80mmol / L, pH7.8 Tris-HCl buffer solution, A 280 Monitor the collected protein peaks and dilute the aliquots for use.

[0029] SM 3+ - Preparation of goat anti-mouse antibo...

Embodiment 2

[0066] Embodiment 2 preparation kit

[0067] Coating plate solid-phase antigen preparation: use 50mmol / L, pH9.6 Na 2 CO 3 -NaHCO 3 The buffer will be OTA-BSA, AFB 1 - Dilute BSA to a final concentration of 10 mg / L as coating solution, add 100 μL to each well of a 48-well microplate, place at 4°C overnight, discard the coating solution, wash three times; add 150 μL of 3 g / L BSA The above-mentioned buffer was blocked and placed overnight at 4°C, the blocking solution was discarded, vacuum-dried, and the strips were sealed and stored at -20°C in a freezer.

[0068] The preparation of reagent: with embodiment 1.

[0069] Reagents provided in the kit:

[0070] The reagents in each box are sufficient for 48 measurements, and the materials in the box are as follows:

[0071] (1).1×48-well plate (4 strips×12 holes, can be split into single holes) coated with OTA-BSA, AFB 1 -BSA.

[0072] (2). 1× buffer solution: 30 mL.

[0073] (3).6×OTA, AFB 1 Standard solution, 1.0mL / bottl...

Embodiment 3

[0083] The reagent provided by the kit is the same as in Example 1, and is used to detect wheat samples.

[0084] The specific detection steps are as follows:

[0085] The wheat sample is processed first: crush the wheat sample to 20 meshes, take 5 grams of the sample and put it in a test tube, add 12.5 mL of the extract (methanol:water=7:3). Stopper and vibrate for 3 minutes, filter, and use Xinhua No. 1 paper as the filter paper. Take 1mL of the filtrate and dilute it with 1mL of distilled or deionized water for later use.

[0086] The fetch package is OTA-BSA and AFB 1 - Microwell coated plate with BSA, add 50 μL of OTA, AFB 1 Add 25 μL of buffer (2) as diluent to dilute OTA antibody 1:10 into the respective microwells, add 25 μL of buffer (2) as diluent and dilute AFB 1:10 1 Antibody, shake at 25-37°C for 0.5-1 hour, wash with washing solution three times; add 50 μL of Eu diluted 1:10 with buffer (2) 3+ - Goat anti-rabbit antibody, 50 μL Sm diluted 1:10 with buffer (2...

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Abstract

A reagent kit for detecting ochratoxin A and aflatoxin B1 at the same time and method thereof are provided, which belongs to fields of time-resolved fluoroimmunoassay. Micro plate is coated with OTA-BSA, AFB1-BSA, adding OTA, AFB1 standard or sample, and then adding OTA monoclonal antibody, AFB1 polyclonal antibody. Dissociated OTA, AFB1 and OTA-BSA, AFB1-BSA on micro plates competing corresponding antibody, and antibody which haven't be connected is eliminated after washing, adding Sm3+-goat anti rat and Eu3+- goat anti rabbit, labeled antibody haven't be connected after immune reaction are eliminated after washing. After adding enhancement solution, detecting Eu and scythe fluorescence intensity cps respectively which are inversely proportional to intensity of OTA and AFB1 in sample respectively by time resolved luminoscope, comparing standard curve and the content of OAT and AFB1 in sample is determined. The reagent kits of present invention has merits of simple structure, convenient operation and low cost, two examining results of OAT and AFB1are obtained by one operation, the present invention is used for detecting of grain, feed and food.

Description

technical field [0001] A Simultaneous Detection of Ochratoxin A and Aflatoxin B 1 The kit and detection method thereof belong to the technical field of time-resolved fluorescent immunoassay (TRFIA), and are used for detecting ochratoxin A (OTA) and aflatoxin B in grain, feed and food 1 (AFB 1 ) content detection. Background technique [0002] Ochratoxin A (OTA) is a toxin produced by the fungus Aspergillus ochraceous and several Penicillium fungi. OTA has been proven to cause damage to the kidneys of animals and humans, and is also a carcinogen. Most of the poisoning caused by mycotoxins is caused by mold-contaminated grains, oil crops, and fermented foods, and mycotoxin poisoning is often manifested in obvious places The clinical manifestations are more complex, including acute poisoning, chronic poisoning, carcinogenicity, teratogenicity and mutagenicity. OTA can be isolated from most grains, including barley, wheat, oats, corn, coffee beans, etc., and poultry fed with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/577
Inventor 黄飚马智鸿张艺朱岚张珏金坚
Owner JIANGSU INST OF NUCLEAR MEDICINE
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