Aflatoxin enzyme-linked immunosorbent assay (ELISA)
- This ELISA test can be done in class as a demonstration or a desktop exercise, if gloves and proper hazardous waste disposal are available. It is a competitive-direct ELISA test that can analyze a sample of corn meal, peanut butter, or another food to determine if the sample is at or below the legal 20 parts per billion (ppb) limit. The test requires no special equipment and can be completed in less than 15 minutes.
- The kits can be purchased and stored in the refrigerator. The contents can be pipetted into microfuge tubes for use by students or student groups. Food samples can be weighed and placed in plastic containers with tops for use with the methanol for aflatoxin extraction.
- Source of kits: Agri-Screen for Aflatoxin, kit #8010, Neogen Corp.
Mycotoxins are poisons produced by fungi. They are commonly found in stored grains and seeds, such as wheat, corn, and peanuts. The fungi that produce the most significant mycotoxins in food are in the genera Aspergillus, Fusarium, and Penicillium. These mycotoxins cannot be detected by smell or taste after the grains or seeds have been processed, but they remain toxic. Toxic effects to animals include hemorrhage, liver damage, cancer, and abortion. In the United States, the legal level of aflatoxin—a mycotoxin produced by Aspergillus flavus—is restricted to 20 ppb.
It is often desirable to run field tests for specific mycotoxins in harvested grain. If the mycotoxin levels appear high, more samples can be sent to a laboratory for additional testing before the grain is sold for human or animal food.
We will test samples of peanuts and corn meal to determine if they exceed the legal limit for aflatoxin. This immunoassay for aflatoxin is a competitive direct (CD) ELISA.
First, the microwells are coated with antibodies specific for aflatoxin. A sample of peanut butter or corn meal is mixed with methanol to extract the aflatoxin. The extract is filtered and added to the test well. Control aflatoxin at 20 ppb (the legal limit) is added to the control well. This is used for comparison with the sample that has an unknown amount of aflatoxin. The enzyme conjugate to which aflatoxin has been bound is added to both wells, where it will compete with any aflatoxin in the sample or with the added control aflatoxin (20 ppb) for binding sites. After incubation, the wells are washed to remove all unbound materials. An enzyme substrate that changes to a blue color is then added to the wells. The more aflatoxin in the sample, the less blue or more red (stop solution) the results become, because the sample aflatoxin will outcompete the aflatoxin bound to the conjugate, causing the color change for binding sites.
Note the differences between the commonly used DAS-ELISA and the CD-ELISA in this figure:
Instructions for the Aflaxtoxin ELISA
Note: Wear gloves for this activity
Sample Preparation and Extraction
- Add 25 milliliters (mL) of 70% methanol to the 5 gram (g) sample in the extraction bottle, and shake vigorously for 3 minutes. The sample has already been weighed for you. Allow the sediment to settle.
- Filter 5 mL of the upper liquid layer using the filter syringe. Collect the filtrate as the sample into the small tube provided with the syringe.
- The sample is now ready for testing. If the sample is not used immediately, cap it to prevent evaporation.
Note: Accurate Pipetting is Ipmortant for this Exercise
Identify all materials before starting. One lab partner should read and cross off each step as the other lab partner performs the step.
- Remove two red-marked mixing wells from the foil packet. Mark one well C for “control,” and mark one well S for “sample.” Place the wells firmly into the well holder. Replace the remaining wells in each packet and reseal.
- Remove two antibody-coated wells from their foil packets. Label one antibody-coated well C for “control,” and label the other S for “sample.” Place the wells firmly into the well holder. Replace remaining wells in each packet and reseal to protect the antibody.
- Using a new pipettor tip, add 75 microliters (μL) of enzyme conjugate from the blue tube to each red-marked mixing well. Discard the tip.
- Using a new tip, add 75 μL of the control (aflatoxin) from the yellow tube to the C red-marked mixing well. Thoroughly mix by inserting the tip into the liquid and pipetting up and down five times. Discard the tip.
- Using a new tip, add 75 μL of the sample extract to the S red-marked well. Thoroughly mix by inserting the tip into the liquid and pipetting up and down five times. Discard the tip.
- Using a new tip, transfer 100 μL from the C red-marked well to the C antibody-coated well (step 2 in the CD ELISA figure).
- Using a new tip, transfer 100 μL from the S red-marked well to the S antibody-coated well (step 2 in the CD ELISA figure).
- Discard the red-marked wells.
- Mix by sliding the antibody-coated wells back and forth on a flat surface in a manner to ensure adequate mixing for 10–20 seconds, without splashing the reagents. Wait 2 minutes after mixing.
- The initial reaction has been completed. Shake out the contents of the wells in the liquid waste container. Fill each well with distilled water and shake out. Repeat five times. Remove all water droplets by turning the wells upside down and vigorously tapping them on an absorbent towel (step 3 in the CD ELISA figure).
- Using a new tip, add 100 μL of enzyme substrate from the green tube to each well. Discard the tip (step 4 in the CD ELISA figure).
- Mix by sliding the wells back and forth on a flat surface for 10–20 seconds, and wait 3 minutes after mixing.
- Using a new tip, add 100 μL of Red Stop from the red tube to each well. Discard the tip. Mix as before.Visually check to confirm thorough mixing.
If the sample well is as blue or darker blue than the control well, the sample contains less than 20 ppb of aflatoxin. If the sample well shows less blue or more red than the control well, the sample contains more than 20 ppb of aflatoxin. For optimal observation of color differences, place the wells on a white surface and read looking down through the solution