SOP: Aflatoxin Screening by ELISA

Aflatoxins from moldy peanuts, corn, and spices are among the most potent natural carcinogens known. ELISA kits give a fast yes-or-no screen before confirmatory testing, letting receivers reject bad lots at the dock.

Chemical analysis converts invisible composition into numbers that specifications, labels, and safety decisions depend on. Consistent technique matters as much as good instruments: small variations in timing, temperature, or reagent preparation shift results enough to fail a batch that should pass, or pass one that should fail.

Purpose

To screen total aflatoxins (B1, B2, G1, G2) in foods by enzyme-linked immunosorbent assay for rapid lot acceptance decisions.

Scope

Applies to peanuts, tree nuts, corn, spices, and dried fruits using a validated commercial ELISA kit. Positive screens above the kit cutoff require confirmatory testing by HPLC or LC-MS.

Equipment and Materials

• Commercial total-aflatoxin ELISA kit (check expiry and storage at 2 to 8 C)
• Microplate reader, 450 nm
• Blender and sample divider
• Methanol and water for extraction (kit-specified ratio, commonly 70 percent methanol)
• Filter paper or centrifuge
• Micropipettes and tips
• Timer

Procedure

1. Grind the entire laboratory sample to pass a 1 mm sieve. Aflatoxin distributes unevenly, so thorough grinding and mixing is critical.
2. Weigh the kit-specified sample amount (commonly 20 g) and extract with the methanol-water mixture by shaking 3 minutes.
3. Filter or centrifuge to obtain a clear extract. Dilute with kit buffer as directed.
4. Bring all kit reagents to room temperature before starting.
5. Pipette standards, controls, and samples into the antibody-coated wells per the kit layout. Run standards in duplicate.
6. Add enzyme conjugate, incubate for the kit-specified time, then wash the plate the specified number of times. Incomplete washing is the top cause of bad results.
7. Add substrate, incubate in the dark, then add stop solution.
8. Read absorbance at 450 nm within 30 minutes of stopping.
9. The assay is competitive: higher aflatoxin gives lower absorbance. Fit the standard curve per kit instructions.
10. Include a kit control with every run; it must fall within the stated range.

Recording Results

Report total aflatoxins in micrograms per kg (ppb) from the standard curve, corrected for dilution. Flag any result above the kit screening cutoff for confirmatory analysis. Record kit lot number and expiry.

Quality Control

Run a reagent blank with every batch and subtract it from sample results. Analyze every sample in duplicate; the relative percent difference between duplicates must not exceed 10 percent for concentrations well above the detection limit. Include a certified reference material or a validated in-house control sample with each batch and plot the result on a Shewhart control chart. Investigate any point outside two standard deviations and reject the batch if a point falls outside three standard deviations. Verify the calibration of volumetric glassware annually against Class A tolerances, and check the analytical balance daily with certified weights. Standardize titrants monthly and label with the expiry date. Document all quality control results in the batch record; sample results must not be reported when associated QC fails.

Troubleshooting

• Poor standard curve: reagents not at room temperature or pipetting errors. Equilibrate fully and check pipette calibration.
• High background: insufficient washing. Wash the specified times with full well volumes and tap dry.
• Erratic duplicates: incomplete sample grinding or mixing. Re-grind and re-extract.
• Matrix interference: some spices suppress the signal. Use the kit-specified dilution for difficult matrices.

Safety Notes

• Aflatoxin standards are highly toxic carcinogens. Handle with gloves in the fume hood; never mouth-pipette.
• Methanol is toxic and flammable. Use in ventilated areas away from ignition.
• Decontaminate spills of standards with bleach solution and dispose as hazardous waste.