🥜 Overview

Peanut allergies are serious — and Ara h 1, a 66 kDa peanut protein, is one of the major culprits behind allergic reactions. This study developed highly sensitive and selective immunoassays — specifically ELISA and lateral flow immunoassay (LFIA) — to detect Ara h 1 in foods and prevent contamination or mislabeling during production.

🧬 1. Introduction

• Ara h 1 is a 7S vicilin storage globulin — part of the cupin superfamily, forming a stable trimer.
• Found abundantly in peanut cotyledons, it’s the main allergen for > 90% of people allergic to peanuts.
• Cross-reactivity with tree nuts exists but is limited because Ara h 1’s sequence is unique.
• Detecting Ara h 1 helps prevent cross-contact and recalls in food manufacturing.

🧫 2. Materials & Methods

🐭 2.1 Immunization

• Four Balb/cByJ mice were injected with purified Ara h 1 mixed with Freund’s adjuvant.
• After several boosts, their spleens were collected to harvest immune B cells.

🧪 2.2 Cell Fusions

• Hybridoma technology fused splenocytes with SP2/0 myeloma cells using PEG.
• Cultures were grown in selective HAT medium until antibody-producing colonies formed.

🧫 2.3 Hybridoma Screening

• Screening via ELISA identified cells producing anti-Ara h 1 antibodies.
• Positive clones were sub-cloned (limiting dilution) to ensure monoclonality and stored in liquid nitrogen.

💉 2.4 Monoclonal Antibodies (MAbs)

• Antibodies were purified via protein-G chromatography and confirmed as IgG1-κ.
• Two top performers: 4A8 (capture) and 13A4 (detection).

🧫 2.5 ELISA Setup

• Direct, indirect, and sandwich ELISAs (sELISA) were tested.
• sELISA (4A8 + 13A4) gave the best results:
  • Sensitivity = 10 ng/mL
  • Log-linear range: 20 μg/mL → 0.05 μg/mL

🌰 2.6 Sample Prep

• Peanut and tree-nut meals were defatted with hexane, proteins extracted in buffer.
• Recombinant Ara h 1 and Ara h 2 were used as controls.

🧴 2.7 Western Blot

• Verified that MAbs bind only to Ara h 1 (~64 kDa), not Ara h 2 (~17 kDa).
• Binding persisted after heat denaturation, showing robust recognition.

🔬 2.8 Epitope Mapping

• Used 40 overlapping peptides of Ara h 1 (18 aa each).
• 4A8 bound peptides 18, 29, 33, 34; 13A4 bound 15, 28, 29.
• Mapping on Ara h 1’s 3D model revealed distinct surface-exposed epitopes.

📏 2.9 LFIA Construction

• Assembled test strips with:
  • 4A8 MAb → test line (T)
  • anti-mouse IgG → control line (C)
  • 13A4-gold nanoparticle conjugate as reporter
• Assembled on nitrocellulose and optimized for flow and stability.

📸 2.10 LFIA Analysis

• Tested with Cube Reader (525 nm).
• Detection limit = 0.5 µg/mL for peanut meal.
• Test positive if both T and C lines visible.

📊 3. Results & Discussion

🌟 3.1 Sensitive Peanut Detection (sELISA)

• 4A8 + 13A4 sELISA achieved 10 ng/mL sensitivity.
• Superior to commercial assays (often ≥ 100 ng/mL).
• Quantitative, reproducible, and highly specific.

🌰 3.2 Specificity

• Six peanut varieties (Argentina, South Africa, USA, China) all tested positive regardless of roasting.
• No signal from tree nuts or soybeans → no cross-reactivity.

🔍 3.3 MAb Binding Confirmation

• sELISA and Western blot verified exclusive Ara h 1 recognition.
• No binding to Ara h 2 or other vicilins.
• Stable even after heating.

🧩 3.4 Epitope Characterization

• Both MAbs bind distinct, surface-exposed epitopes on Ara h 1.
• Epitopes survive denaturation → great for robust tests!
• Ensures non-overlapping binding ideal for sandwich assays.

🧷 3.5 Rapid LFIA Detection

• LFIA produced visible color lines in 5 min.
• Quantified digitally:
  • LOD = 0.5 µg/mL for peanut meal
  • LOD = 10 ng/mL for pure Ara h 1
• No false positives with Ara h 2 or tree nuts.
• Outperformed commercial kits in both speed and specificity.

✅ 4. Conclusion

• Developed two monoclonal antibodies (4A8 + 13A4) specific to Ara h 1.
• Created:
  • A sensitive sELISA (LOD = 10 ng/mL)
  • A fast LFIA strip (LOD = 0.5 µg/mL, 5 min readout)
• Both assays resist heat effects, detect all peanut types, and show zero cross-reactivity.
• Useful for food industry allergen monitoring, consumer safety, and regulatory testing.

🧠 Key Takeaways

⚙️ Real-World Impact

These assays can:

• Prevent accidental allergen exposure 😷
• Reduce product recalls 🚫
• Enable quick on-site food safety checks 🧾
• Support global allergen monitoring programs