Zearalenone-vomitoxin double-channel immune quantitative test strip

A technology for zearalenone and vomitoxin, which is used in immunoassays, measuring devices, biological tests, etc., can solve the problem that it is not suitable for rapid detection of grain mycotoxins, expensive equipment, and the lack of simultaneous detection of vomitoxin and zearalenone. Immunoquantitative test strips and other issues, to achieve the effect of solving market needs, high accuracy and small errors

Inactive Publication Date: 2019-11-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the method for simultaneous detection of mycotoxins is mainly HPLC-MS, but due to the requirements of expensive equipment and complicated sample pretreatment, it is not suitable for rapid detection of mycotoxins in grains, so it is urgent to develop Simultaneous detection of multiple mycotoxins
Although there are reports on test strips for the detection of vomitoxin at present, such as the rapid detection card with application numbers 201720645327.3 and 201710319370.5, the fluorescent immunochromatography kit of 201720357308.0, the colloidal gold detection card of 20162005179

Method used

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  • Zearalenone-vomitoxin double-channel immune quantitative test strip
  • Zearalenone-vomitoxin double-channel immune quantitative test strip
  • Zearalenone-vomitoxin double-channel immune quantitative test strip

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0048] Example 1 Preparation of fluorescent probes for labeling zearalenone monoclonal antibodies and vomitoxin monoclonal antibodies with Eu-fluorescent microspheres

[0049] The specific preparation method is as follows:

[0050] (1) Take 50μL (1% solid content) of carboxyl fluorescent microspheres (purchased from Xiamen Nordao Technology, particle size 300nm) placed at 4℃, ultrasonically disperse, and add 800-1000μL of 0.05M 2-(N- Morpholine) ethanesulfonic acid (MES, C 6 H 13 NO 4 S·H 2 O) Activation buffer, centrifuge at 14500rpm for 10-15min (the temperature is controlled at about 15℃ during centrifugation);

[0051] (2) Discard the supernatant, add 600-800μL of MES buffer, resuspend in ultrasound, and repeat centrifugation for 2-3 times;

[0052] (3) Discard the supernatant, add 200μL of MES buffer to ultrasonic resuspend, add 50μL of 10mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, C 8 H 17 N 3 ·HCl), 50μL 10mg / mL N-hydroxysuccinimide (NHS, C 4 H 5 NO ...

Example Embodiment

[0059] Example 2 Assembly of zearalenone-vomitin dual-channel immunoassay strips based on labeled fluorescent microspheres

[0060] The structure diagram of the Zearalenone-Vomitoxin immune test strip is as follows image 3 As shown, from left to right on the bottom plate are sample pads, nitrocellulose (NC) membrane and absorbent paper. The key to the assembly of test strips is to ensure consistent transferability between the parts. The sample pads are stacked on NC On the film, the two overlap by about 5mm. Similarly, the absorbent paper is stacked on the NC film, and the two overlap by about 5mm. Use a strip cutter to cut the glued board into test strips about 4mm wide, with a plastic base and a card shell Assemble, sealed and stored at 4°C for later use.

[0061] The assembly method is as follows: spray zearalenone complete antigen (0.2mg / mL) and vomitin complete antigen (0.3mg / mL) on the nitrocellulose membrane as the detection line (T1 line) and detection line (T2 line), Spr...

Example Embodiment

[0062] Example 3 Drawing of standard curve based on zearalenone-vomitin immunoassay strips labeled with fluorescent microspheres

[0063] The drawing method of the standard curve is:

[0064] The Eu-fluorescent microspheres prepared in Example 1 were labeled with zearalenone monoclonal antibody (Eu-ZEN-mAb) and vomitin monoclonal antibody (Eu-DON-mAb) as fluorescent probes, and 40 μL Mixed standard solution of zearalenone and vomitoxin, 50μLEu-ZEN-mAb solution and 50μLEu-DON-mAb solution are mixed uniformly, and 140μL of the mixture is slowly dropped into the test strip sample pad, and chromatographed at 37°C for 10min. Then record the T value and C value through the HG-98 immunoquantitative analyzer. The concentration gradients (ZEN / DON) of the mixed standard of zearalenone and vomitoxin are: 1 / 1μg / L, 2 / 2μg / L, 4 / 5μg / L, 6 / 10μg / L, 8 / 20μg / L , 10 / 25μg / L. Take the logarithm of the concentration of each standard as the abscissa, and take T / T 0 The (inhibition rate) value is the ordin...

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Abstract

The invention discloses a zearalenone-vomitoxin double-channel immune quantitative test strip, and belongs to the technical field of immunoassay and rapid detection. A fluorescent probe is prepared bymarking fluorescent microspheres; a fluorescent microsphere-zearalenone monoclonal antibody, a fluorescent microsphere-vomitoxin monoclonal antibody and a fluorescent microsphere-goat anti-rabbit secondary antibody are included, and zearalenone artificial antigen, fluorescent microsphere artificial antigen and goat anti-mouse secondary antibody are sprayed on a nitrocellulose membrane to serve asa detection line T1, a detection line T2 and a quality control line C respectively to prepare the immunochromatographic test strip; and zearalenone and vomitoxin in the sample are analzyed simultaneously and quantitatively by reading the fluorescence value of the detection lines in a fluorescence immunoassay analyzer by means of a competitive immunoassay method. The method overcomes the defect that colloidal gold is not easy to store in the test strip technology, and the method for preparing the fluorescent probe is simple, efficient and high in sensitivity.

Description

technical field [0001] The invention relates to a zearalenone-vomitin dual-channel immunological quantitative test strip, which belongs to the technical field of rapid detection of immune analysis. Background technique [0002] Zearalenone (ZEN), also known as F-2 toxin, is an estrogen-like mycotoxin produced by Fusarium graminearum, Fusarium three-line and Fusarium moniliforme, etc. It is widely found in corn, wheat, Sorghum and other grains and their products have strong reproductive and developmental toxicity and teratogenic effects, which can lead to slow growth and immunosuppression of poultry and livestock, and can enter the human body through the food chain, causing blood and immune toxicity, and have carcinogenic activity It can cause tumors and bring great harm to human health. [0003] Deoxynivalenol, deoxynivalenol (DON), is one of the trichothecene olefins. As a common mycotoxin, its main pollutants are wheat, barley, oats, corn and other grain crops. [0004]...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/558G01N33/577G01N33/543G01N33/533
CPCG01N33/582G01N33/54313G01N33/558G01N33/577G01N33/533G01N2469/20G01N2333/37G01N33/56961G01N33/54388
Inventor 孙秀兰李淼王海鸣刘莹纪剑张银志孙嘉笛皮付伟
Owner JIANGNAN UNIV
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