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Reagent box for detecting aftatoxin B and detecting method thereof

A kind of technology of aflatoxin and reagent kit, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complicated operation, expensive instruments and equipment, low sensitivity, etc., and achieve the effect of convenient use, simple structure and high sensitivity

Inactive Publication Date: 2005-09-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aflatoxin B 1 There are multiple assay methods, such as: thin layer chromatography TLC (sensitivity is 5 μg / kg), high performance liquid chromatography HPLC (sensitivity is 0.02 μg / kg), immunofluorescence staining (sensitivity is 0.025 μg / ml), but Due to the shortcomings of time-consuming, low sensitivity, expensive equipment, complicated operation and not suitable for the detection of large quantities of samples, it has been phased out

Method used

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  • Reagent box for detecting aftatoxin B and detecting method thereof
  • Reagent box for detecting aftatoxin B and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1 prepares kit and detects corn sample

[0014] AFB 1 - Preparation of KLH antigen

[0015] 1. Dissolve 2mg of AFB in dimethylformamide (DMF) 1 ;

[0016] 2. Use 0.13mol / L NaHCO 3 Dissolve 2mg of KLH carrier protein in coupling buffer;

[0017] 3. Take 0.8mg AFB 1 The solution is added to the solution of KLH;

[0018] 4. Dissolve 4 mg of carbodiimide (EDC) in double distilled water, and add 100 μL to the above mixture, and let it act for 2 hours at room temperature (protect from light);

[0019] 5. After centrifugation, take the supernatant and separate it on a column (Sephadex G-25) for UV scanning detection.

[0020] polyclonal aflatoxin B 1 Antibody preparation:

[0021] 1. Choose a 4-week-old healthy New Zealand white rabbit weighing about 1.5Kg. AFB 1 is a hapten that will AFB 1 Linked with BSA as an antigen.

[0022] 2. Preparation of water-in-oil antigen emulsifier: Mix 2mg of AFB with 1.2ml of Freund's complete adjuvant or Freund's incomp...

Embodiment 2

[0056] Embodiment 2 prepares kit

[0057] polyclonal aflatoxin B 1 Preparation of antibody: same as Example 1.

[0058] Eu 3+ - Preparation of goat anti-rabbit antibody:

[0059] Take 1ml of 5g / L goat anti-rabbit antibody dissolved in 50mmol / L PBS pH7.0, change the buffer condition through PD-10 column, and the eluent is 50mmol / L NaCl containing 0.155mol / L NaCl 2 CO 3 -NaHCO 3 pH9.0 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 and add Eu containing 0.2mg 3+ -N 2 -[p-isocyanate-benzyl]-diethylenetriaminetetraacetic acid (Eu 3+ -DTTA) in a vial with magnetic stirring at 28°C for 16 hours. The reaction solution was chromatographed on a Sephadex-G50 column (1×40 cm) equilibrated with 80 mmol / LTris-HCl pH 7.8 buffer solution to collect protein peaks, and diluted for use.

[0060] Preparation of coated plate solid-phase antigen: sa...

Embodiment 3

[0075] Embodiment 3 prepares kit

[0076] AFB 1 -The preparation of KLH antigen is the same as in Example 1.

[0077] polyclonal aflatoxin B 1 The preparation of antibody is the same as in Example 1, omitted.

[0078] Eu 3+ - Preparation of goat anti-rabbit antibody:

[0079] Take 2ml of 5g / L goat anti-rabbit antibody dissolved in 50mmol / L PBS pH7.0, change the buffer condition through PD-10 column, and 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 1000μl and add Eu containing 0.4mg 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 / LTris-HCl pH7.8 buffer solution, A 280 ...

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Abstract

The aflatoxin B1 detecting kit and its detecting method belongs to the field of time-resolved fluorescent immunoassay (TRFIA) technology, and is used in the detection of aflatoxin B1 (AFB1) content in food and feed. The kit of the present invention detects AFB1 in TRFIA based on marker immune reaction. The microporous board has coated AFB1-HRP, added AFB1 standard or sample, and added AFB1 antibody. The free AFB1 and the AFB1-HRP in the microporous board compete AFB1 antibody, un-connected AFB1 antibody is washed off, EU3+ sheep anti-rabbit antibody is added and un-connected EU3+ sheep anti-rabbit antibody after marker immune reaction is washed off. After reinforcing solution is added, the fluorescence strength cps, which is proportional inversely to AFB1 concentration in the sample, is determined with time-resolved fluorescent instrument to determine the AFB1 content in the tested sample via comparison with standard curve.

Description

technical field [0001] A method for detecting 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 aflatoxin B in grain, feed and food thereof 1 (referred to as AFB 1 ) content detection. Background technique [0002] Aflatoxin B 1 (AFB 1 ) is a group of highly toxic secondary metabolites produced by the fungi Hspergillus flavus and A. parasilicus strains, especially AFB 1 It is a strong pollutant with a wide distribution range, which can cause acute poisoning death of humans and various feed animals, and can also cause carcinogenicity, teratogenicity, and mutagenicity. Even the content of tens of μg / kg is still very toxic. More than 1mg / kg of aflatoxin in food and feed is highly toxic. Its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. After eating food seriously contaminated by aflatoxin, fever, abdominal pain, vomiting, and loss of ...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/543G01N33/569
Inventor 陶文沂金坚黄飚赵晓联张莲芬时瑾
Owner JIANGNAN UNIV
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