1. Sample preparation
– Sample grinding: Food samples must be ground finely to ensure homogeneity.
– Weighing: Weigh an amount of sample according to the ELISA kit instructions (usually 5-25g, depending on the sample type and the mycotoxin to be tested).
– Extraction: The sample is extracted with extraction solution (usually methanol: water or acetonitrile: water at a specific percentage ratio). For example, for aflatoxin, methanol: water (70:30) is typically used.
– Shaking or stirring: To ensure that mycotoxins are extracted from the sample, the solution must be stirred or shaken evenly for a specified time (usually 10-30 minutes).
– Filtering or centrifuging: After extraction, the mixture must be filtered or centrifuged to remove particulates and sediment. The filtrate will be used for the next step.
2. Sample dilution
Dilute the extracted sample with the dilution buffer supplied by R-Biopharm, following the specific instructions for each ELISA kit. This step adjusts the mycotoxin concentration into the measurable range.
3. ELISA kit preparation
– Preparing the ELISA plate: The R-Biopharm ELISA plate is already coated with antibodies specific to the mycotoxin to be tested (for example: Aflatoxin, Ochratoxin, Fumonisin, Zearalenone, T-2/HT-2 toxin).
– Adding standard solutions: Prepare mycotoxin standard solutions of known concentration and add them to separate wells of the ELISA plate to create the standard curve.
– Adding the extracted sample: Add the diluted sample to the remaining wells on the ELISA plate.
4. Sample incubation
– After adding the standard solutions and samples, the ELISA plate is incubated for a set period (usually 30 minutes to 1 hour) at room temperature or in an incubator (depending on the kit instructions).
– During this process, the antibodies in the wells will bind to any mycotoxin present in the sample (if present).
5. Plate washing
After incubation, the ELISA plate is washed with wash buffer to remove substances not bound to the antibodies. Washing is performed several times (usually 3-5 times) to ensure complete removal of contaminants.
6. Enzyme and substrate addition
– Adding the enzyme-conjugated secondary antibody: Add the conjugate enzyme (for example peroxidase enzyme) to the wells. This enzyme will bind to the mycotoxin-antibody complex.
– Further incubation: The ELISA plate is incubated again to allow the enzyme reaction to take place (usually 15-30 minutes).
– Adding the substrate: After incubation, add substrate solution (chromogen/substrate) to the wells. The substrate will react with the enzyme and produce color (usually blue).
7. Stopping the reaction
Add stop solution: After a set incubation time, the stop solution (usually sulfuric acid or similar) is added to the wells to stop the enzyme reaction. The color will change from blue to yellow.
8. Reading the results
– Reading optical density (OD): Use an ELISA reader to measure the optical density (OD) at the appropriate wavelength (usually 450 nm).
– Comparison with the standard curve: Compare the OD value of the sample with the standard curve built from the standard solutions to determine the mycotoxin concentration in the sample.
9. Analysis and reporting
– Calculating the mycotoxin concentration: Based on the OD results, calculate the mycotoxin concentration in the sample by comparison with the standard curve.
– Reporting the results: Prepare a results report and compare it with the permissible safety limits for mycotoxins in food and feed.
* Notes when performing the ELISA technique:
– Follow the instructions for each ELISA kit exactly, because incubation times and solutions may differ.
– Ensure that laboratory equipment and glassware are thoroughly cleaned to avoid cross-contamination.
– Each R-Biopharm kit has a more detailed procedure and requires following the specifications supplied by the manufacturer.
The ELISA technique from R-Biopharm stands out with its high sensitivity, allowing detection of low mycotoxin concentrations, and can be applied to many different types of food, from cereals and grains to dairy and meat products. Please contact us for advice and supply of products suitable for your production process!