Qualitative and quantitative immunoassay method of vomitoxin DON direct competitive chemiluminescence

A technology of chemiluminescence immunity and vomitoxin, which is applied in chemiluminescence/bioluminescence, analysis by making materials undergo chemical reactions, and analysis of materials can solve the problems of interference affecting experimental results, inaccurate detection, and rarely used. To achieve the effect of improving detection efficiency, improving sensitivity, improving detection efficiency and detection sensitivity

Inactive Publication Date: 2019-07-19
YANTAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this kind of detection is extremely inaccurate, and it is very susceptible to the interference of various factors to affect the experimental results, resulting in false positive or false negative results, so it is rarely used at present.

Method used

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  • Qualitative and quantitative immunoassay method of vomitoxin DON direct competitive chemiluminescence
  • Qualitative and quantitative immunoassay method of vomitoxin DON direct competitive chemiluminescence
  • Qualitative and quantitative immunoassay method of vomitoxin DON direct competitive chemiluminescence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] In this embodiment, the working curve and detection limit of vomitoxin DON are determined, and the working curve equation is calculated through this embodiment.

[0087] The working curve equation is established as follows:

[0088] Take blank grains as 9 blank samples, homogenize the samples, add 10mL PBS solution respectively, shake and vortex extract for 5min, centrifuge at 9000g at 4°C for 10min, take the supernatant and pass it through a 0.45μm water phase filter membrane to obtain the blank matrix solution.

[0089] Blank matrix solution was used to configure blank sample DON matrix spiked standards with concentrations of 1, 4, 10, 40, 100, 400, 1000 and 4000 ng / mL, and the remaining 1 part was used as DON-free 0 standard solution, do 3 parallels for each concentration, to obtain the matrix spiked standard solution of the test substance.

[0090] Determination method: Use a micropipette to extract 50 μL of the test solution of each concentration and add it to th...

Embodiment 2

[0095] accuracy test

[0096] Before the test, firstly select the standard substance of vomitoxin DON, then dissolve it with acetonitrile and shake it up to prepare the standard solution of vomitoxin DON.

[0097] The preparation of need testing solution: select the non-polluting cereal food as blank sample, then sample is homogenized, add a certain amount of DON standard solution in the blank cereal food (corn, wheat), make its concentration in the cereal respectively 0.5, 1, 1.5μg / g, set five parallels for each concentration, mix and let stand for 30min, then 10mL PBS solution respectively, shake and vortex extract for 5min, centrifuge at 4℃, 9000g for 10min, take the supernatant and pass through 0.45μm The aqueous phase was filtered to obtain the test solution.

[0098] Determination method: Use a micropipette to extract 50 μL of the test solution of each concentration, add it to the enzyme-labeled plate coated with vomitoxin DON, add the enzyme-labeled antibody prepared i...

Embodiment 3

[0105] cross reactivity test

[0106] Nine toxins with similar structures and functions to DON were selected to determine the cross-reactivity rate. The 50% inhibitory concentration of each toxin was obtained through the standard curve. The ratio of this method to other similar cross-reactivity was calculated using the following formula.

[0107] Cross-reaction rate (%) = (50% inhibition of deoxynivalenol DON concentration / 50% inhibition of deoxynivalenol DON analog concentration) × 100%

[0108] The experiment was repeated 3 times, and the results were averaged.

[0109] Table 3 Specificity of DON detection

[0110]

[0111] Experimental results show that the chemiluminescence detection method developed by the present invention can only recognize vomitoxin DON and its metabolites DOM-1 and 3-AcDON, while the cross-reaction to other analogues including 15-AcDON is negligible. The antibody used in the invention has better broad specificity and can be used for rapid det...

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Abstract

The invention relates to a method for qualitatively and quantitatively detecting vomitoxin DON in cereals by directly competing chemiluminescence immunity. The detection method comprises the steps ofpreparing enzyme-labeled vomitoxin DON antibody, directly competing chemiluminescence immunity detection, establishing of a working curve equation and quantitatively detecting vomitoxin DON of a grainsample to be detected. According to an improved sodium periodate method in the method, a saturated NaBH4 methanol solution is adopted to carry out reduction reaction, the defects that the NaBH4 aqueous solution is prone to oxidizing and cannot be stored for a long time are overcome, so that the limitation of the current preparation of the solution is avoided; a direct competitive chemiluminescence immunity detection technology improves the detection efficiency and sensitivity; a highly sensitive luminol chemiluminescence substrate is adopted to replace a traditional TMB substrate, and thus the reaction sensitivity is greatly improved; by establishing the working curve, the concentration of vomitoxin DON in the sample can be quickly and accurately obtained, the complexity of detection andcalculation is reduced, and the method is applicable to a wide range of grassroots detection work.

Description

technical field [0001] The invention relates to the technical field of rapid detection of food safety, in particular to a method for qualitative and quantitative detection of vomitoxin DON by direct competitive chemiluminescence immunoassay. Background technique [0002] With the improvement of living standards, people have higher and higher requirements for food safety, and the food safety problems caused by mycotoxin contamination in grains have become the focus of attention all over the world. Cereal foods have complex components, low concentrations of analytes, and usually large sampling volumes, which put forward higher requirements for the sensitivity and rapidity of analytical methods. Direct chemiluminescent enzyme-linked immunosorbent assay (D-CLIA) technology combines highly sensitive chemiluminescent technology with immune reaction, which overcomes the stability of traditional enzyme labeling technology and indirect competitive reaction operation Complicated and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/535G01N21/76
CPCG01N21/76G01N33/535G01N33/581
Inventor 李彦伸袁智鹏毛馨尤艳莉
Owner YANTAI UNIV
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