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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 201620051794.9, in addition, the test for the detection of zearalenone There are also reports on paper strips, such as the fluorescent quantitative rapid detection test strips with application numbers 201720453761.1 and 20210307321.7, but there is a lack of immunoquantitative test strips for simultaneous detection of vomitoxin and zearalenone. Therefore, this patent uses time-resolved fluorescent microspheres as Tracer, a dual-channel immunochromatographic assay for the simultaneous detection of zearalenone and deoxynivalenol in grains was established based on the immunocompetitive reaction model

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

Embodiment 1

[0048] Example 1 Preparation of fluorescent probes for zearalenone monoclonal antibody and vomitoxin monoclonal antibody respectively labeled with Eu-fluorescent microspheres

[0049] The specific preparation method is as follows:

[0050] (1) Take 50 μL (1% solids content) of carboxyl fluorescent microspheres (purchased from Xiamen Nodao Technology Co., Ltd., particle size 300 nm) placed at 4°C, ultrasonically disperse them, and add 800-1000 μL of 2-(N- Morpholine) ethanesulfonic acid (MES, C 6 h 13 NO 4 S·H 2 0) Activation buffer, centrifuged at 14500rpm for 10-15min (the temperature is controlled at about 15°C during centrifugation);

[0051] (2) Discard the supernatant, add 600-800 μL MES buffer, resuspend by ultrasonication, repeat centrifugation and wash 2-3 times;

[0052] (3) Discard the supernatant, add 200 μL MES buffer for ultrasonic resuspension, add 50 μL 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, C 8 h 17 N 3 HCl), 50 μL 10...

Embodiment 2

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

[0060] The structure diagram of the zearalenone-vomitoxin immune test strip is as follows image 3 As shown, there are sample pads, nitrocellulose (NC) membrane and absorbent paper from left to right on the bottom plate. The key to test strip assembly is to ensure consistent transmissibility between each part. The sample pad is stacked on the NC. On the membrane, the two overlap by about 5mm. Similarly, the absorbent paper is stacked on the NC membrane, and the two overlap by about 5mm. Use a strip cutter to cut the pasted board into test strips about 4mm wide. Assemble and store sealed at 4°C for later use.

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

Embodiment 3

[0062] Example 3 Drawing of Standard Curve Based on Zearalenone-Deoxynivalenol Immunization Test Strips Labeled Fluorescent Microspheres

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

[0064] Zearalenone monoclonal antibody (Eu-ZEN-mAb) and vomitoxin monoclonal antibody (Eu-DON-mAb) respectively labeled with the Eu-fluorescent microspheres prepared in Example 1 were used as fluorescent probes, and 40 μL Mix zearalenone and vomitoxin standard solution, 50 μL of Eu-ZEN-mAb solution and 50 μL of Eu-DON-mAb solution, and slowly drop 140 μL of the mixture into the test strip sample pad, and perform chromatography at 37°C for 10 minutes. Then record T value and C value by HG-98 immunological quantitative analyzer. The concentration gradients (ZEN / DON) of zearalenone and vomitoxin mixed standard are: 1 / 1μg / L, 2 / 2μg / L, 4 / 5μg / L, 6 / 10μg / L, 8 / 20μg / L , 10 / 25 μg / L. Take the logarithmic value of each standard concentration as the abscissa, and use T / T 0 The (inhibition rate) val...

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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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