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Direct competitive chemiluminiscence qualitative and quantitative immunoassay for T-2 toxins

A chemiluminescence immunoassay and quantitative analysis technology is applied in the field of direct competitive chemiluminescence immunoassay for qualitative and quantitative detection of T-2 toxin, which can solve the problems of interference affecting experimental results, inaccurate detection, and seldom use, etc., to improve detection efficiency and improve Sensitivity and detection time reduction effect

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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  • Direct competitive chemiluminiscence qualitative and quantitative immunoassay for T-2 toxins
  • Direct competitive chemiluminiscence qualitative and quantitative immunoassay for T-2 toxins
  • Direct competitive chemiluminiscence qualitative and quantitative immunoassay for T-2 toxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] This embodiment measures the T-2 toxin working curve and detection limit, and calculates the working curve equation through this embodiment.

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

[0088] Take 13 blank grains as blank samples, homogenize the samples, add 10mL methanol / PBS (60 / 40, v / v) solution respectively, shake and vortex extract for 10min, centrifuge at 9000g at 4°C for 5min, take 5mL The supernatant was diluted with 25 mL of PBS, and passed through a 0.45 μm aqueous phase filter to obtain a blank matrix solution.

[0089] The blank matrix solution was used to configure the blank sample T-2 toxin matrix spiked standard with a series concentration, the initial concentration was 10000ng / mL, and three-fold dilutions were made successively, a total of 11 concentrations, the lowest concentration was 0.169ng / mL, and the remaining 1 part was used as the For the 0 standard solution containing T-2 toxin, do 3 parallels for each concentration to obtai...

Embodiment 2

[0095] accuracy test

[0096] Before the test, first select the T-2 toxin standard product, then dissolve it with acetonitrile and shake it up to prepare the T-2 toxin standard solution.

[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 T-2 toxin standard solution in the blank cereal food (corn, wheat), make it in the cereal The concentrations were 40, 200, and 1000 μg / kg, respectively, and five parallels were set for each concentration, mixed and allowed to stand for 30 minutes, and then 10 mL of methanol / PBS (60 / 40, v / v) solution was added to each sample, shaken , Extract by vortexing for 10 min, centrifuge at 9000g at 4°C for 5 min, take 5 mL of the supernatant and add 25 mL of PBS to dilute, pass through a 0.45 μm aqueous phase filter to obtain the solution to be tested.

[0098] Determination method: Use a micropipette to extract 50 μL of the test solution of each c...

Embodiment 3

[0105] cross reactivity test

[0106] Nine toxins with similar structures and functions to T-2 toxin 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-reactivity rate (%)=(inhibit 50% T-2 toxin concentration / inhibit 50% T-2 toxin analog concentration)×100%

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

[0109] Table 3T-2 Specificity of toxin detection

[0110]

[0111] Experimental results show that the chemiluminescence detection method developed by the present invention can only recognize T-2 toxin and its metabolite HT-2, while the cross-reaction to other toxin analogs can be ignored. The antibody used in the invention has better broad specificity and can be used for rapid detection of T-2 toxin and HT-2 toxin in cereal food.

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Abstract

The invention relates to a method for qualitatively or quantitatively detecting T-2 toxins in cereals by means of direct competitive chemiluminiscence immunoassay. The detection method comprises the steps of preparing an enzyme-labeled T-2 toxin antibody, performing direct competitive chemiluminiscence immunodetection, establishing a working curve equation and performing quantitative detection ofthe T-2 toxins in a grain sample to be tested. An improved sodium periodate method of the invention adopts a saturated NaBH4 methanol solution for reduction reaction, thereby overcoming the disadvantage that the NaBH4 aqueous solution is easily oxidized and cannot be stored for a long time, and avoiding the limitation that the solution is instantly used once prepared; the direct competitive chemiluminescence immunodetection technology improves the detection efficiency and the detection sensitivity; a high-sensitivity Luminol substrate is adopted to replace a traditional TMB (tetramethylbenzidine) substrate, so that the reaction sensitivity is greatly improved; and the concentration of the T-2 toxins in the sample can be rapidly and accurately obtained by establishing a working curve, so that the detection and calculation complexity is reduced, and the method is suitable for the wide application of basic-level 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 T-2 toxin 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 immunosorbentassay (D-CLIA) technology combines high-sensitivity chemiluminescent technology with immune reaction, which overcomes the stability of traditional enzyme labeling technology and indirect competitive reaction It has disadvantages such as co...

Claims

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Application Information

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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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