Triplex lateral flow immunoassay for sensitive and simultaneous detection of allergenic proteins from walnut, almond and hazelnut
This study presents a validated triplex lateral flow immunoassay capable of the rapid, sensitive, and simultaneous detection of walnut, almond, and hazelnut proteins in complex food matrices, thereby supporting effective precautionary allergen labeling in the food industry.
Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer
For millions of people around the world, a simple meal can carry a hidden threat. A single bite of a cookie or a sip of a drink might contain a trace of an ingredient that their immune system recognizes as a dangerous invader. This reaction, known as an allergy, can range from mild discomfort to a life-threatening emergency called anaphylaxis, where the body's systems shut down rapidly. Among the most dangerous triggers are tree nuts, such as almonds, hazelnuts, and walnuts. Because these nuts are so common in processed foods and are often used in facilities that handle many different products, the risk of accidental cross-contamination is high. To protect consumers, food manufacturers rely on strict labeling, but they also need a way to verify that their products are truly free of these specific nuts before they reach the shelf. This is where rapid testing comes in, offering a quick check to ensure safety without the long wait times of traditional laboratory analysis.
Scientists at the Universidad de Zaragoza and their partners have developed a new tool to meet this need: a single, portable test strip capable of detecting three different types of tree nuts at the same time. This device, known as a multiplex lateral flow immunoassay, works much like a home pregnancy test but is designed to hunt for specific proteins found in almonds, hazelnuts, and walnuts. Instead of requiring three separate tests, each with its own chemicals and waiting period, this new strip allows a food safety inspector to dip a single sample into a buffer solution and place it on the device. Within minutes, the strip reveals whether any of the three nuts are present. The researchers built this tool by training antibodies, which are proteins that act like highly specific locks, to recognize the unique keys found only in the proteins of these three nuts. When the antibodies find their match, they trigger a visible red line on the strip, signaling a positive result.
The team did not just build the device; they put it through a rigorous series of checks to ensure it works reliably in the real world. They tested the strip against a vast array of foods, including fruits, vegetables, grains, meats, and other seeds, to make sure it would not give a false alarm. The results showed that the test is extremely selective; it only lights up for the three target nuts and ignores everything else, including other types of nuts like pecans or Brazil nuts, which are chemically similar but distinct. The researchers also checked how sensitive the device is, determining the smallest amount of nut protein it could reliably find. They found that the strip could detect as little as one microgram of almond protein per gram of food, half a microgram for walnut, and two and a half micrograms for hazelnut in raw extracts. These levels are low enough to catch even tiny traces of contamination that could harm a sensitive consumer.
However, food is rarely just raw ingredients; it is cooked, baked, and mixed with fats and acids, all of which can change how proteins behave. To see if the test could handle these changes, the team baked cookies that contained tiny, known amounts of ground almonds, hazelnuts, and walnuts. They then tested these baked goods with their new strip. Even after the high heat of the oven, which can alter the structure of proteins, the device successfully identified the nuts. In these baked cookies, the strip could still detect two micrograms of almond, one and a half micrograms of walnut, and twelve micrograms of hazelnut per gram of food. The researchers also tested the strip in complex liquid and solid foods like chocolate drinks, fresh cheese, and herbal liqueurs, confirming that the test works accurately regardless of the food's texture or acidity.
A common problem with some rapid tests is that if a sample contains too much of the target substance, the test can actually fail to show a result, a phenomenon known as the hook effect. The researchers checked for this by testing samples with extremely high concentrations of nut protein. They found that while the test lines did become fainter at very high levels, the device remained reliable for the low concentrations that matter most for safety. The concentrations required to cause this failure were so high that they would only occur if someone intentionally added a large amount of nuts as an ingredient, in which case the product would already be required to carry a warning label. This means the test is safe for its intended purpose: finding accidental traces.
The study concludes that this new strip is a powerful addition to the food industry's safety toolkit. By allowing for the simultaneous detection of three major allergens in a single, fast, and inexpensive test, it helps manufacturers make better decisions about their products. If a factory knows exactly which nuts are present and in what amounts, they can use precautionary labeling more responsibly, avoiding the overuse of warnings that can confuse consumers while still protecting those with severe allergies. The device has been validated to work in both raw ingredients and finished, cooked foods, offering a practical way to manage the risk of tree nut allergies in an increasingly complex food supply.
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