🥜 Food Allergens (ELISA)
In short
Quantitative determination of food allergens (gluten, milk, egg, peanuts, soy etc. The test is performed according to AOAC 2012.01 / AOAC 2014.03 / AOAC 991.19 (seria metod ELISA); Determination range: 5–200 mg/kg (typical calibration curve range). The procedure comprises 11 steps (about 1 h 40 min–2 h 30 min in total); it is used for: Quality control of gluten-free production (limit < 20 mg/kg according to Codex), Verification of production line cleaning (swabs) — prevention of cross-contamination, Testing of raw materials and semi-finished products for undeclared allergens.
At a glance
- Standard: AOAC 2012.01 / AOAC 2014.03 / AOAC 991.19 (seria metod ELISA)
- Category: Food Chemistry
- Procedure steps: 11
- Total time of stages: 1 h 40 min–2 h 30 min
- Limit of detection (LOD): 1–5 mg/kg (ppm) of allergen
- Determination range: 5–200 mg/kg (typical calibration curve range)
- Analysis time: 30–90 min (depending on kit — FAST vs standard)
Overview
Determination of food allergens is a key element of food safety — Regulation (EU) 1169/2011 requires declaration of 14 major allergens on food product labels. The ELISA (Enzyme-Linked Immunosorbent Assay) method is the most widely used laboratory technique for quantitative determination of allergenic proteins in food and contact surfaces.
The method principle is based on specific antigen–antibody binding. Two main formats are available: sandwich ELISA — detects intact proteins and large fragments with two epitopes, and competitive ELISA — also detects small protein fragments, which is crucial for highly thermally processed food.
Most commonly used commercial kits are: RIDASCREEN (R-Biopharm), Veratox and Reveal (Neogen), AgraQuant (Romer Labs) and ELISA Systems. These kits have AOAC-OMA (Official Methods of Analysis) validation and are recommended by Codex Alimentarius. Typical determined allergens are: gluten (wheat, rye, barley prolamins), milk proteins (casein, β-lactoglobulin), egg proteins (ovalbumin, ovomucoid), peanut proteins (Ara h1, Ara h2), soy, nuts, sesame and mustard proteins.
ELISA detection limits are typically 1–5 mg/kg (ppm) of allergen in the product, allowing assessment of compliance with safety thresholds. Laboratories performing these tests participate in PT programs (FAPAS, LGC) and are accredited according to ISO 17025.
Method principle
In sandwich ELISA: microplate wells are coated with antibodies specific for a given allergen. Food sample extract is incubated in the well — allergen binds to antibodies. After washing, second enzyme-labeled antibody (HRP or AP) is added, which binds to another epitope of the allergen (forming a "sandwich"). After re-washing, enzyme substrate is added — a colored product is formed, whose intensity (absorbance) is proportional to allergen concentration. Reading on microplate reader at 450 nm.
Applications
- Quality control of gluten-free production (limit < 20 mg/kg according to Codex)
- Verification of production line cleaning (swabs) — prevention of cross-contamination
- Testing of raw materials and semi-finished products for undeclared allergens
- Control of imported food by Sanitary Inspection
- Food testing in the context of product recalls
- Validation of cleaning procedures in food plants
- Analysis of infant and young child food (strict allergen regime)
Key parameters
| Parameter | Value |
|---|---|
| Limit of detection (LOD) | 1–5 mg/kg (ppm) of allergen |
| Determination range | 5–200 mg/kg (typical calibration curve range) |
| Analysis time | 30–90 min (depending on kit — FAST vs standard) |
| Format | 96-well microplate (12 strips × 8 wells) |
| Reading wavelength | 450 nm (with reference 620–650 nm) |
| Gluten limit according to Codex | < 20 mg/kg gliadin (= < 20 ppm gluten) |
Standard
- Standard number
- AOAC 2012.01 / AOAC 2014.03 / AOAC 991.19 (seria metod ELISA)
- Title (PL)
- Oznaczanie alergenów pokarmowych metodą immunoenzymatyczną ELISA
- Title (EN)
- Determination of food allergens by enzyme-linked immunosorbent assay (ELISA)
Step-by-step procedure
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Sample preparation
Homogenize food sample to obtain uniform mass. Weigh 1 g into extraction tube.
-
Extraction
Add 10 mL extraction buffer from kit. Shake/mix for 10 min at room temperature (or 60°C for gluten with Mendez cocktail).
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Centrifugation and dilution
Centrifuge 10 min at 2500 × g. Collect supernatant. Dilute according to kit instructions (typically 1:10 or 1:100).
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Application on microplate
Apply 100 µL of calibration standards (5 concentrations + zero), samples and controls to appropriate wells. Each in duplicate.
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Incubation I
Incubate microplate 30 min at room temperature (or 10 min for FAST kits). Gentle shaking.
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Washing
Wash 3× with 300 µL washing buffer. Remove residual liquid by inverting and blotting on absorbent paper.
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Incubation with conjugate
Add 100 µL of antibody-enzyme solution (conjugate). Incubate 30 min RT.
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Washing II
Wash 3× as in step 6.
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Color detection
Add 100 µL enzyme substrate (TMB). Incubate 15 min in darkness. Add 100 µL stop solution.
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Absorbance reading
Measure absorbance on microplate reader at 450 nm (reference 620 nm). Perform within 30 min of stopping.
-
Results calculation
Plot calibration curve (absorbance vs concentration). Read allergen concentration in samples. Convert to mg/kg in product.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Microplate reader | Tecan Sunrise, BioTek ELx800, Thermo Multiskan FC | 15 000–50 000 PLN |
| Incubator (37°C or RT) | Microplate shaking incubator, Heidolph Titramax 1000 | 3 000–8 000 PLN |
| Microplate washer | BioTek ELx50, Tecan HydroFlex, Thermo Wellwash | 15 000–40 000 PLN |
| Homogenizer | IKA Ultra-Turrax T25, Stomacher 400 | 5 000–15 000 PLN |
| Multichannel pipettes | Eppendorf Research Plus 8-channel (10–100 µL, 100–1200 µL) | 2 000–5 000 PLN |
| Analytical balance | Mettler Toledo MS105DU, RADWAG AS 220.R2 | 8 000–20 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| ELISA gluten kit (RIDASCREEN Gliadin) | — | R-Biopharm R7001 — sandwich ELISA for gliadin determination, AOAC-OMA certificate, 96 tests, ~2 500 PLN |
| ELISA milk kit (RIDASCREEN FAST Milk) | — | R-Biopharm R4652 — FAST 30 min, LOD 0.7 mg/kg, 48 tests |
| ELISA egg kit (Neogen Veratox Egg) | — | Neogen 8470 — sandwich ELISA, LOD 2.5 mg/kg, 48 tests |
| ELISA peanuts kit (RIDASCREEN FAST Peanut) | — | R-Biopharm R6802 — LOD 0.1 mg/kg, AOAC-OMA validation |
| ELISA soy kit (AgraQuant Soy) | — | Romer Labs — competitive ELISA for soy proteins, 96 tests |
| Extraction buffer | — | Supplied in kit or according to manufacturer's instructions (e.g. Mendez cocktail for gluten) |
Health and safety (OHS)
- Stop solution (H₂SO₄) — corrosive, use glasses and gloves
- Extraction buffer — may contain mercaptoethanol (toxic) — fume hood
- TMB substrate — suspected mutagenic effect, use gloves
- Biological material (food samples with allergens) — risk of reactions in sensitized persons
- Microplate waste — disposal as laboratory waste
Frequently asked questions
Which standard describes this test?
The test is performed according to AOAC 2012.01 / AOAC 2014.03 / AOAC 991.19 (seria metod ELISA) — “Determination of food allergens by enzyme-linked immunosorbent assay (ELISA)”.
How does this method work?
In sandwich ELISA: microplate wells are coated with antibodies specific for a given allergen. Food sample extract is incubated in the well — allergen binds to antibodies.
What is the measuring range and accuracy?
Limit of detection (LOD): 1–5 mg/kg (ppm) of allergen; Determination range: 5–200 mg/kg (typical calibration curve range); Analysis time: 30–90 min (depending on kit — FAST vs standard); Format: 96-well microplate (12 strips × 8 wells).
How long does the test take?
The times given for the individual stages add up to approximately 1 h 40 min–2 h 30 min. The procedure comprises 11 steps.
What equipment is required?
Microplate reader, Incubator (37°C or RT), Microplate washer, Homogenizer, Multichannel pipettes, Analytical balance. Indicative cost of the core instrument (Microplate reader): 15 000–50 000 PLN.
Where is this test used?
Quality control of gluten-free production (limit < 20 mg/kg according to Codex); Verification of production line cleaning (swabs) — prevention of cross-contamination; Testing of raw materials and semi-finished products for undeclared allergens; Control of imported food by Sanitary Inspection; Food testing in the context of product recalls; Validation of cleaning procedures in food plants; Analysis of infant and young child food (strict allergen regime).
What safety precautions apply?
Stop solution (H₂SO₄) — corrosive, use glasses and gloves; Extraction buffer — may contain mercaptoethanol (toxic) — fume hood; TMB substrate — suspected mutagenic effect, use gloves; Biological material (food samples with allergens) — risk of reactions in sensitized persons; Microplate waste — disposal as laboratory waste.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number AOAC 2012.01 (seria).