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Food Allergy Testing

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FPIES: Not As Delicious As It Sounds

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From time to time, we like to write about the rarer forms of food allergy. We’ve covered  Eosinophilic EsophagitisOral Allergy Syndrome, and allergies to red meat and water! Today’s blog topic will cover another lesser-known, but very serious food allergy: Food Protein-Induced Enterocolitis Syndrome (FPIES for short). 

What is it?

FPIES is a non-IgE immune system reaction to food that affects the gastrointestinal (GI) tract. IgE stands for the antibody immunoglobulin E, and most allergic reactions (think top eight most common food allergies) involve this antibody. FPIES is cell-mediated, which results in a delayed allergic reaction.

Notably, unlike typical food allergies, FPIES does not show up on standard allergy tests.

Who does it affect?

FPIES reactions often show up in the first weeks or months of a child’s life. Sometimes the child may be a little bit older if they’ve been exclusively breastfed. First reactions often occur when introducing solid foods, such as infant formulas or cereals, which are typically made with dairy or soy.

What are the common trigger foods?

For infants that experience FPIES from solid foods, rice and oats are the most common triggers. Other reported triggers include, but are not limited to: milk, soy, barley, sweet potato, squash, green beans, peas, and poultry. 

Any food protein can be a trigger and some infants may be sensitive to other foods as well. As with any food allergy, some children may only react to 1-2 foods, while others may react to several. 

What are the symptoms?

FPIES can cause severe symptoms following ingestion of a trigger food. Classic FPIES symptoms include diarrhea, severe vomiting, and dehydration. These can lead to changes in body temperature, blood pressure, and lethargy. Upon ingestion of a trigger food, there is a characteristic delay of 2-3 hours before the onset of symptoms. 

Symptoms can range from mild (such as an increase in reflux and several days of runny stools) to life-threatening (shock). In several cases, after repeated vomiting, children often begin to vomit bile. Diarrhea typically follows and can last up to several days. It’s important to note that each child is unique and may experience their own range and severity of symptoms. 

Importantly, many infants who are eventually diagnosed with FPIES are initially suspected to have a severe infection or sepsis based on their symptoms. 

How is it diagnosed?

FPIES cannot be detected with traditional allergy testing methods, such as skin prick or blood tests that measures IgE antibodies. It is accordingly tough to diagnose.

Researchers are currently looking to atopy patch testing (APT) for its effectiveness in diagnosing FPIES. APT involves placing the trigger food in a metal cap, which is left on the skin for around 48 hours. The skin is then observed for symptoms in the days following removal.

Additionally, the outcome of APT may determine if the child is a potential candidate for an oral food challenge: the gold standard for food allergy diagnosis. A medical doctor, often an allergist and/or gastroenterologist, should be involved in the diagnosis of FPIES.

Is there a silver lining?

The good news is that FPIES usually resolves with time! Many children outgrow FPIES by age 3, allowing kids to introduce the offending foods back into their diet over time. With proper medical attention and a personalized dietary plan, children with FPIES can grow and thrive! 

- Meg and the Allergy Amulet Team

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A Brief History of Molecularly Imprinted Polymers: The Heart of Allergy Amulet’s Technology

 Dr. Joseph BelBruno, Scientific Advisor at Allergy Amulet.

Dr. Joseph BelBruno, Scientific Advisor at Allergy Amulet.

If you’ve been following our blog for a while, you’ve probably heard us talk about our scientific approach to detecting food allergens: molecularly imprinted polymer (MIP) films. These films lie at the technological heart of the Allergy Amulet. 

In a previous post, we covered the basics of how the technology works. Quick review: an MIP is a polymer (plastic) formed in the presence of target template molecules to create molecular molds. Once the templates are removed from our films, they leave behind trillions upon trillions of plastic molded “locks” that bind when in the presence of our molecular “keys.” In our films, these keys represent allergenic ingredients.

Today’s post looks at the history of MIP technology. While our science team has made considerable breakthroughs in the MIP field, we didn’t pull the foundation technology out of thin air. In fact, the body of work on MIPs dates back almost 100 years!

The first scientific mention of MIPs was back in 1931: a scientist named M. V. Polyakov discovered that when he made polymers out of silica in the presence of another molecule, the polymers would selectively absorb that molecule.

Some say the origins of MIP technology started with Jean Dickey at Cal Tech. He tried imprinting silica with organics back in 1949. The modern approach to imprinting began in Europe in the seventies and eighties with Klaus Mosbach in Sweden, Günter Wulff in Germany, Borje Sellegren in Amsterdam, and Karsten Haupt in France. These and other scientists developed many of the classic methods for creating imprinted polymers. Importantly, some studies found that in select circumstances, MIPs could imitate the functions of receptors, enzymes, and other biological molecules. 

After these initial discoveries, scientists identified new applications throughout the 20th century—mostly in drug separation—and you can currently buy MIP resins from leading science manufacturers like Sigma Aldrich. Yet, as with any nascent technology, development progressed slowly. Only in the past ten years have MIPs finally hit their stride: nearly half of all MIP papers were written in the past decade. Over time, researchers have optimized the conditions and techniques for developing MIPs, which has expanded the types of molecules MIPs are capable of imprinting. This has dovetailed with advancements in nanotechnology and communications technology. For example, the nanomaterials we use in our sensors were prohibitively expensive ten years ago. With these advancements, diverse commercial applications of MIP technology are finally becoming a reality.

For a detection device, successfully imprinting an allergenic ingredient (or any other target) is only half the battle.

Why is that? Well, even if an MIP can selectively bind the target molecule, it does so on a nanoscopic scale—we would have no way of knowing that binding occurred. Creating an MIP sensor accordingly requires a system that can translate that imprinting into something comprehensible (what scientists would call a transducer). While there has been significant research into different types of transducers for MIPs, a common approach has been electrical conductivity: an easily measured property that is already widely used in sensor technology.

There are several methods that convert successful imprinting to an electrical response. Two of the most popular methods involve using either conductive polymers or combining the MIPs with conductive nanomaterials.

Some of the first successes in creating conductive MIPs originated with Dr. Joseph BelBruno: a world-renowned chemist, Dartmouth chemistry professor, and Scientific Advisor at Allergy Amulet. Dr. BelBruno’s research laid the foundation for developing conductive MIP sensors. His work spawned the first commercial application of MIP sensors.

Allergy Amulet is positioned to become the second company to commercialize MIP sensors, and the first to create MIP sensors for detecting allergenic ingredients. Our sensors combine MIPs with an electrical response to create sensors for detecting molecular tracers of allergenic ingredients in food.

As with any novel application of an existing technology, it is important to recognize and pay tribute to the work of those before us. Learning and studying from the successes and failures of our predecessors is how we will advance as a scientific community, and advance as a society.    

-      Nazir and the Allergy Amulet Team

 

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Get Your Geek On: The Science Behind Food Allergy Testing

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Food testing is serious business. It’s also a large and growing one: the market for food testing kits was valued at $1.58 billion in 2016. That figure is expected to climb to $2.38 billion by 2022.

Since the enactment of the Food Safety Modernization Act (FSMA) of 2011, food manufacturers are increasingly implementing comprehensive food testing procedures. Allergen testing has accordingly taken on a more prominent role in food safety plans. Traditionally, food allergen testing has been confined to the lab; but as new technologies emerge, and old technologies evolve, that’s starting to change.

In this post, we break down the most common food allergen detection technologies. We also discuss emerging technologies and approaches (including ours!) and why changes in food allergen detection are on the horizon. Spoiler alert: prepare for some major geeking out!

Liquid Chromatography-Mass Spectrometry (LC-MS)

As its name implies, liquid chromatography-mass spectrometry (LC-MS) is a two-phase test. During the liquid chromatography phase, a food sample is dissolved in a liquid and funneled through a highly-pressurized chromatography column, which separates molecules based on size and structure.

The mass spectrometer measures the mass of each molecule, as well as the masses of any molecular fragments. A molecule’s mass and fragmentation pattern provide identifying information about the molecule.

Caffeine: Mass Spectrum

   Mass spectrum fingerprint of caffeine.

Mass spectrum fingerprint of caffeine.

Although LC-MS is a highly-selective tool for molecular identification, LC-MS instruments are expensive and large. Even modest instruments can cost tens of thousands of dollars and stand as high and wide as a microwave. Higher-end instruments can be as large as a car! Test times are also relatively long, ranging from 10 to 30 minutes per food sample. Accordingly, these tests are generally confined to lab environments at present.

Ultraviolet, Visible Light, Infrared, and Raman Spectroscopy

These spectroscopic methods rely on light absorption. A molecule’s chemical structure determines which light wavelengths may be absorbed and the degree of absorption. Spectrometers shine a range of wavelengths at a food sample, and a molecule’s relative absorption of those different wavelengths generates an identifying “fingerprint” for that molecule. You can think of spectroscopy as the enLIGHTened approach to molecular detection 😉.

Caffeine: Infrared Spectrum

    Infrared   spectral fingerprint for caffeine. Peaks and dips signify   degree     of molecular light absorption.

Infrared spectral fingerprint for caffeine. Peaks and dips signify degree of molecular light absorption.

Spectral fingerprints are ideal for identifying molecules in samples containing only a few ingredients. Spectra can be generated in a span of seconds, with high-resolution versions taking only one to two minutes. However, identifying molecules in complex mixtures like food samples can present serious challenges for spectroscopic methods, as spectral fingerprints are likely to overlap, making individual molecules difficult or impossible to identify—especially in low quantities. Accordingly, spectroscopy does not currently lend itself to allergen detection in food samples. Moreover, any spectrometer that could potentially afford sufficient selectivity for allergen detection would be large and costly.

Immunoassays & ELISA

Immunoassay tests rely on antibodies. Antibodies are naturally-occurring proteins in the body’s immune system designed to recognize and fight potentially harmful foreign materials. Each antibody is formed to recognize a specific target—usually a protein or protein fragment. Since the 1950’s, scientists have cultivated antibodies to function outside of the body. These antibodies led to tests known as immunoassays. There are many variants of immunoassays, including ELISA (enzyme-linked immunosorbent assay) tests, which many food manufacturers use to test for allergens during the manufacturing process.

In a typical immunoassay, a liquid sample suspected of containing a particular allergenic protein is exposed to a test strip containing antibodies, which are formulated to recognize that specific protein. If the target protein is present, the protein will stick to the antibodies on the test strip and a secondary reaction will stain the bound protein, causing the test strip to change color.

Immunoassays are highly selective, portable, and can produce results in as little as a few minutes. However, culturing and harvesting specific antibodies can be expensive. Moreover, antibodies—like most proteins—are sensitive to harsh conditions like high temperatures or extreme pH levels. The integrity of these tests, therefore, depends on adequate storage conditions. Antibodies are also known to have relatively short shelf lives and typically degrade within one year.

PCR and Molecular Beacons

Another technology in the allergen detection field involves identifying DNA sequences from an allergenic ingredient using a combination of a polymerase chain reaction (PCR) and molecular beacons. Don’t worry, it’s not as complicated as it sounds.

One way to test for an allergenic ingredient is to detect DNA segments unique to that ingredient. DNA is made of two complementary strands, and when one strand finds its complement, they bind. Simple enough. PCR uses the complementary nature of DNA to identify and exponentially replicate target DNA strands. This replication makes the DNA strands easier to detect using what are called molecular beacons: specialized molecular tags that turn fluorescent upon binding to a target DNA strand. These illuminated beacons can then be measured with a fluorescence spectrometer. While PCR-based assays are sensitive and selective, these tests are generally better suited for laboratory environments because they require automated laboratory equipment.

Historically, molecular beacons have been used to detect nucleotide chains like DNA; more recently, molecular beacons are being used to bind and stain proteins–including allergens–instead of DNA sequences. In this approach, PCR is not necessary, as the molecular beacons attach directly to the protein. Notably, molecular beacon tags require a fluorescence spectrometer to measure the target allergenic protein or nucleotide sequences.

Molecularly Imprinted Polymers (Allergy Amulet’s Technology!)

Molecularly imprinted polymer (MIP) sensors are an exciting emerging technology. MIPs are highly-specialized plastic films molded to recognize a single target molecule, such as an allergenic protein or a chemical tracer for an allergenic ingredient. Historically, molecularly imprinted polymers have been used for drug separation and delivery. Only recently have MIPs been adapted for use as molecular recognition elements in electronic sensing devices.

Building an MIP is similar in concept to creating a lock for which the target molecule is the key. Our polymer films contain hundreds of trillions of cavities (locks), which recognize a specific target molecule (key) by size, shape, and complimentary electron charge distribution. The molding procedures used for MIPs mean that they can be designed to target a wide variety of molecular targets. Our Scientific Advisor, Dr. Joseph BelBruno, was the first to develop electronic MIP sensors for detecting nicotine and marijuana. Allergy Amulet is the first to develop MIP sensors for detecting allergenic ingredients.

   
  
   
  
    
  
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      Imprinted cavity molded to bind to a specific target molecule.

Imprinted cavity molded to bind to a specific target molecule.

Because the core ingredient in a MIP-based sensor is a specialized plastic, MIP films are highly durable and affordable to produce. The high specificity of target binding, coupled with a straightforward electrochemical resistance measurement, allows for rapid and portable testing.

That’s it! Now you know the science behind allergen detection methodologies. We hope you enjoyed geeking out with us for a short while. Until next time!

-        The Allergy Amulet Science Team

 

These scientific explanations have been simplified to accommodate our nontechnical readership. 

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