Time-resolved fluoroimmunoassay kit for detecting fumonisins B1 and detection method thereof

A technology of time-resolved fluorescence and fumonisins, which is used in analytical materials, fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of expensive equipment, complicated operation, low sensitivity, etc., and achieves simple structure, convenient use and sensitivity. high effect

Inactive Publication Date: 2010-04-28
JIANGSU INST OF NUCLEAR MEDICINE
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] There are multiple assay methods for fumonisin B1 at present, such as: thin layer chromatography TLC (sensitivity is 500ng / g), high performance liquid chromatography HPLC (sensitivity is 80ng / g), liquid mass spectrometry (sensitivity 100ng / g) / g), but due to time-consuming, low sensitivity, expensive instruments and equipment, complicated operation and not suitable for large-scale sample detection, etc., its popularization and application are limited.
Enzyme-linked immunoassay (ELISA) (with a sensitivity of 5ng / g) has been paid more and more attention by people because of its strong specificity, high sensitivity, easy operation, no need to directly contact toxins, and it is especially suitable for the detection of large batches of samples. Adopted, but the sensitivity of detection and the stability of reagents are still difficult to meet the requirements

Method used

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  • Time-resolved fluoroimmunoassay kit for detecting fumonisins B1 and detection method thereof
  • Time-resolved fluoroimmunoassay kit for detecting fumonisins B1 and detection method thereof
  • Time-resolved fluoroimmunoassay kit for detecting fumonisins B1 and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 Preparation of kit and detection of corn samples

[0020] The reagents provided in the kit are enough for 96 measurements, and the materials in the box are as follows:

[0021] (1). 1 x 96-well plate (8 strips x 12 wells, which can be split into single wells) coated with FB1-BSA.

[0022] (2). 6×FB1 standard solution, 1.0mL / bottle, the concentration of the standard solution is: 0, 1.0, 10, 100, 500, 1000ng / mL.

[0023] (3). 1× FB1 monoclonal antibody freeze-dried product, dissolved in 0.5mL distilled water.

[0024] (4).1×Eu 3+ - Lyophilized goat anti-mouse antibody, dissolved in 0.5mL distilled water.

[0025] (5). 1× enhancement solution: 15mL.

[0026] (6). 1×washing solution: 30mL, dilute with distilled water 1:25 when used.

[0027] (7). 1× buffer solution: 30 mL.

[0028] Reagents that should be prepared by the laboratory

[0029] Methanol.

[0030] 70% Methanol Solution: Prepare 70% methanol solution by mixing 30mL distilled or deionized water a...

Embodiment 2

[0044] The reagents provided in the kit are enough for 48 measurements, and the coated plate in the box is: 1×48 well plate (4 strips×12 wells, which can be split into single wells) coated with FB1-BSA. The remaining reagents provided by the kit are the same as in Example 1 and are used to detect wheat samples. The specific detection steps are as follows:

[0045] The wheat sample is firstly processed: crush the wheat sample to 20 mesh, take 5 grams of the crushed wheat sample and put it in a test tube, add 25 mL of 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 9mL of distilled or deionized water for later use.

[0046] Take the FB1-BSA strip, add 50 μL of FB1 standard or processed samples to their respective microwells, add 50 μL of FB1 monoclonal antibody diluted 1:20 in buffer, shake at 25°C for 1 hour, wash with washing solution 3 times, Add 1:20 dilute...

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Abstract

The invention discloses a time-resolved fluorescence immunoassay kit for detecting fumonisins B1 and a detection method thereof, belonging to the technical field of time-resolved fluoroimmunoassay (TRFIA) for detecting the content of FB1 in grain, fodder and food. The prepared kit adopts the TRFIA to detect the FB1. A micropore plate is coated with FB1-BSA, is added with FB1 standard or sample, and is added with FB1 monoclonal antibody. Dissociative FB1 and the FB1-BSA on the micropore plate compete for the FB1 monoclonal antibody, unconnected FB1 monoclonal antibody is removed by means of washing, Eu3+ -sheep anti-mouse antibody is added, and the unconnected Eu3+ -sheep anti-mouse antibody is removed by means of washing after labeled immune reaction. After adding strengthening liquid, fluorescence intensity (cps) is detected by a time-resolved fluoroimmunoassay apparatus, wherein the fluorescence intensity is in inverse proportion to the concentration of the FB1, and the content in the FB1 can be ensured by contrasting a standard curve. The kit has simple structure, convenient use, low price and high sensitivity more than 0.05ng/mL.

Description

technical field [0001] A time-resolved fluorescent immunoassay kit for detecting fumonisin B1 (FB1) and a detection method thereof belong to the technical field of time-resolved fluorescent immunoassay (TRFIA), and are used for detecting FB1 content in grain, feed and food. Background technique [0002] Fumonisins are a group of new mycotoxins mainly produced by Fusarium moniliforme first reported internationally in 1988. They mainly contaminate grains, reproduce and metabolize in the growth substrate, and produce a variety of mycotoxins. So far, 11 structural analogues have been found, among which fumonisin B 1 (FB 1 ) is the main component of fumonisins, accounting for about 70% of the total toxins, and is the main cause of the toxic effects of fumonisins. The total fumonisin content in moldy corn can be as high as 52.670mg / kg. In addition, there are also pollution situations in foods and feeds such as rice, wheat, beer, and milk. Fumonisins can cause equine leukomalac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/543G01N21/64
Inventor 黄飚张珏王柯周彬陈蕴金坚
Owner JIANGSU INST OF NUCLEAR MEDICINE
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