ELISA Experimental Procedure for Detecting Aflatoxin B1 in Peanuts

Biotoxins in food, such as aflatoxins, bacterial toxins, and shellfish toxins, are significant risk factors in the global food safety landscape. Aflatoxin B1, a highly carcinogenic mycotoxin, is primarily found in nuts and grains and poses a threat to human liver health. This article aims to provide a standardized and reference-worthy ELISA experimental procedure to help laboratories quickly and accurately detect Aflatoxin B1 in peanut samples, offering technical support for food safety monitoring and compliance management in enterprises.

Figure 1. ELISA principle based on antibody–enzyme detection

👉As part of routine food safety control, aflatoxin testing is commonly included in comprehensive food biotoxin detection programs provided by professional third-party laboratories, such as Alfa Chemistry's Biotoxin Detection Services.

Experimental Objective and Scope

The objective is to detect the content of Aflatoxin B1 (AFB1) in peanut samples using the ELISA method, achieving rapid, sensitive, and reproducible quantitative analysis, and to evaluate the results against FDA/USDA food safety limits in the United States.

Materials, Reagents, and Equipment

Sample: Dried peanut powder (particle size<0.5 mm)

Extraction solution: 70% methanol-water (v/v)

ELISA kit: Aflatoxin B1-specific, suitable for the US market

Buffers and reagents: PBS buffer, wash solution, enzyme substrate, stop solution

Equipment: Microplate, pipettes, and tips

Centrifuge: ≥4000 rpm

Optional: 0.45 μm filter membrane or immunoaffinity column

Peanut Sample Preparation and Extraction Procedure

1. Grinding the sample: Grind the peanut sample into a uniform powder (particle size<0.5 mm).

2. Solvent extraction

• Weigh 5 g of the sample and add 50 mL of 70% methanol-water (1:10 w/v).
• Shake for 30 minutes or perform ultrasonication for 15 minutes to fully release AFB1.

3. Centrifugation: Centrifuge at 4000 rpm for 10 minutes to remove solid impurities.

4. Filtration: Filter the supernatant through a 0.45 μm filter membrane; optionally, purify with an immunoaffinity column.

5. Dilution: Dilute the extract 2-fold according to the kit instructions before loading onto the ELISA plate.

ELISA Assay Procedure for Aflatoxin B1 Analysis

1. Plate Loading: Add 100 μL of sample or standard to each well. Include positive controls, negative controls, and replicate samples.

2. Incubation: Incubate at room temperature for 30 minutes (or as specified by the kit instructions).

3. Washing: Wash the plate 3–5 times to remove unbound substances.

4. Addition of Enzyme-Conjugated Secondary Antibody: Add 100 μL per well and incubate at room temperature for 30 minutes.

5. Second Washing: Wash 3–5 times to reduce background.

6. Substrate Reaction: Add 100 μL of substrate, incubate at 37°C in the dark for 10–15 minutes.

7. Stopping the Reaction: Add 50 μL of stop solution. The color will change from blue to yellow.

8. Absorbance Measurement: Measure the optical density (OD) at 450 nm.

Data Processing, Standard Curve, and Example Results

1. Standard Curve

• Standard concentrations (ng/mL): 0, 2, 5, 10, 20
• Absorbance (OD450): 1.20, 0.95, 0.75, 0.50, 0.35

2. Sample Concentration Calculation and Example Results

3. Quality Control

• Positive control OD: 0.60–0.65
• Negative control OD: 1.15–1.25
• Replicate sample deviation ≤10%, meeting experimental requirements

Quality Control and Precautions

• Thorough sample grinding and complete extraction are key to ensuring accurate results.
• High-fat samples can be purified using immunoaffinity columns to reduce interference.
• Plate washing and pipetting operations should be consistent to ensure reproducibility.
• Perform all operations protected from light to preserve substrate activity.
• If the sample concentration exceeds the assay range, dilute appropriately and re-test.

Frequently Asked Questions (FAQs)