Vomiting Toxin Tester Enzyme-linked immunoassay procedure: Vomiting toxin, also known as deoxynivalenol (DON), is one of the trichothecene olefins, which is usually grown by moldy algae in cereals such as wheat, corn, barley, and valerian. Generated by the prime. Vomiting toxin DON is widely distributed in the world, mainly polluting cereal crops such as wheat, barley, corn, etc. It also pollutes food products. People and animals may have widespread toxic effects after eating food grains contaminated with vomiting toxins. In recent years, it has been found that vomiting toxin DON may be associated with human esophageal cancer and IgA nephropathy, and poses a threat to human and animal health. When humans and animals ingest food/feed contaminated with vomiting toxin DON, acute poisoning symptoms such as anorexia, vomiting, diarrhea, fever, unstable standing, and slow reaction may occur, which may damage the hematopoietic system and cause death. Studies have shown that vomiting toxin DON may have an impact on the immune system, with obvious embryotoxicity and certain teratogenic effects, may be genotoxic, but no carcinogenic and mutagenic effects. Due to the serious harm of vomiting toxin DON, it has attracted widespread attention from all countries. There is a strict limit on the amount of vomiting toxin DON in cereals and feed. The limit of vomiting toxin DON in Chinese grain, pig compound feed, yak compound feed and lactating animal compound feed is 1.0mg/kg, and the limit of vomiting toxin DON in cattle compound feed and poultry compound feed is 5.0mg/kg. 4 equipment and reagents needed 4.1 Instrument: CSY-E96D vomiting toxin detector , homogenizer, oscillator, centrifuge, graduated pipette, balance (sensitivity 0.01g) 4.2 Micropipette: single channel 20μl-200μl, 100μl-1000μl, multi-channel 300μl 5 sample pretreatment 5.1 Notes before sample processing: Laboratory equipment must be clean and use disposable tips to avoid contamination and interference with experimental results. 5.2 Dosing: Solution 1: complex solution The 2× complex solution was diluted 2-fold with deionized water for reconstitution of the sample, and the complex solution was stored for one month at 4 °C. 5.3 Sample pre-processing steps: 5.3.1 Grain (rice, corn, millet, etc.) and feed treatment methods 1) Weigh 2 g of the pulverized sample in a 50 ml centrifuge tube, add 10 ml of deionized water, shake for 5 minutes, and centrifuge at room temperature for 4000 rpm for 10 minutes; 2) Take 0.1ml of the supernatant, add 0.9ml of the complex solution, and mix; 3) Take 50 μl for analysis. Sample dilution factor: 50 detection limit: 150ppb 6 vomiting toxin detector enzyme-linked immunoassay steps Remove the required reagent from the 4 ° C refrigerated environment, and equilibrate at room temperature for more than 30 min. When the washing solution is refrigerated, the crystal may be returned to room temperature to be fully dissolved. Each liquid reagent should be shaken before use. Remove the required number of microplates and frames, place the unused microplates in a ziplock bag and store at 2-8 °C. Before the start of the experiment, the 20X concentrated washing solution was diluted with 20 times of deionized water into a working washing solution. 6.1 No.: The micropores corresponding to the sample and the standard are numbered sequentially, and each sample and the standard are made in parallel with 2 holes, and the position of the standard hole and the sample hole is recorded. 6.2 Loading reaction : Add standard solution or sample 50μl/well to each microwell, then add 50μl/well of antibody working solution, gently shake for 5 seconds, mix and shake at 37°C for 30 minutes. 6.3 Washing : Drain the liquid in the well and wash it thoroughly with 250 μl/well of working washing solution for 5 times at a time. Finally, pat dry with absorbent paper (bubbles that have not been removed after pat drying can be pierced with a clean tip) ). 6.4 Enzyme reaction : 100 μl/well of the enzyme label was added, and the reaction was carried out in the dark at 37 ° C for 30 minutes. 6.5 Washing : Same as above 6.6 Color : Add 50 μl/well of substrate A, add 50 μl/well of substrate B, gently shake for 5 seconds, mix and incubate for 15 minutes at 37 °C. 6.7 End: Add 50 μl/well of stop solution, gently shake and mix to stop the reaction. 6.8 Measured absorbance: The absorbance value per well was measured at 450 nm using a CSY-E96D vomiting toxin detector (double wavelength 450/630 nm is recommended). The assay should be completed within 10 minutes of terminating the reaction. 7 results analysis 7.1 Calculation of percent absorbance The percent absorbance of the standard solution or sample is equal to the average value of the absorbance value of the standard solution or sample (double well) divided by the absorbance value of the first standard solution (0 ppb), multiplied by 100%, ie Percent absorbance value (%) = A ×100% A0 A—the average absorbance value of the standard solution or sample solution Average absorbance value of A0—0ppb standard solution 7.2 Drawing and calculation of standard curve The semi-logarithmic plot of the standard solution is plotted with the percent absorbance of the standard solution as the ordinate and the logarithm of the corresponding standard solution concentration (ppb) as the abscissa. Substituting the percent absorbance of the sample into the standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the corresponding dilution factor is the actual concentration of the analyte in the sample. If the kit professional analysis software is used for calculation, it is more convenient for accurate and rapid analysis of a large number of samples. (Welcome to call) 8 considerations 8.1 Room temperature below 25 ° C or reagents and samples not returned to room temperature (25 ° C) will result in low OD values ​​for all standards. 8.2 If the plate hole is dry during the washing process, the standard curve will not be linear and the repeatability is not good. Therefore, the next step should be taken immediately after the plate is patted dry. 8.3 The mixing should be uniform, the washing should be thorough, and the reproducibility in the ELISA analysis depends largely on the consistency of the washing. 8.4 Seal the microplate with a cover film during all incubations to avoid light exposure. 8.5 Do not use kits that have expired. Do not exchange reagents from different batches. 8.6 If any color of the coloring solution indicates deterioration, it should be discarded. A 0 standard absorbance value of less than 0.5 units (A450nm < 0.5) indicates that the reagent may deteriorate. 8.7 The reaction stop solution is corrosive and avoids contact with the skin. 9 storage and storage period Storage conditions: The kit is stored at 2-8 ° C to avoid freezing. Shelf life: The product is valid for 1 year, and the production date can be found in the box. 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Vomiting toxin detection enzyme-linked immunoassay