The new science of food allergy diagnostics

For the physicians who rely on your diagnostic laboratory, food allergy diagnostics can be perplexing. Consider the teenager who experiences nausea and stomach cramps after eating peanuts. Whole extract specific IgE (s-IgE) testing for peanut demonstrates relatively low sensitization to peanut (1.2kUA/L), although her clinical history links peanut ingestion to symptoms. While this is good information, some critical questions remain unanswered: Is her sensitization specific to peanut, the allergen source being tested? Or to one of its cross-reactive proteins? Should she eliminate all foods containing peanut from her diet? What is her risk of having a systemic reaction? And should she be referred for an oral food challenge (OFC)?

Specific-IgE in vitro blood testing has emerged as a valuable diagnostic tool for allergic conditions when interpreted in the context of a patient’s history and clinical symptoms.¹ Such testing is accurate, well standardized, and supported by National Institutes of Health guidelines for the diagnosis and treatment of food allergy.² Both blood testing and skin prick testing rely on whole allergen extracts that contain a multitude of potential allergenic proteins. Detectible amounts of specific IgE indicate sensitization, but not necessarily clinically relevant allergy. A patient may be sensitized to an allergen, meaning specific IgE is produced, but not allergic, meaning clinical symptoms are not associated with the presence of specific IgE. Similarly, a patient may experience clinical symptoms but have relatively low levels of specific IgE to the source. In the case of peanut, one study showed that 77.6% of sensitized patients may not be at risk for a systemic reaction.³ Now a groundbreaking new development called component-resolved diagnostics (CRD) is refining our understanding of allergic sensitization to individual proteins in an allergen source, to better help clinicians assess the risk of clinically significant reactions or the chances of developing tolerance in their sensitized patients.

The science of component diagnostics

A food source contains dozens of specific proteins, only some of which are allergenic and capable of contributing to clinical disease (Figure 1). Whole extract testing can identify specific IgE binding to one or more of the proteins found in a particular food, but cannot identify to which one(s) the IgE is binding. Antibodies formed to species-specific protein components typically bind only to the proteins particular to that species, but homologous proteins (with similar structures) may also exist in related or unrelated species. Antibodies, formed to these proteins from one species, may also bind to the same, or closely related, structures from other species; this phenomenon is known as cross-reactivity. For example, patients whose test results indicate sensitization to peanut, using whole peanut extract, may actually only be reacting to cross-reactive pollen allergens that share homologous component proteins with peanut. They may not actually be sensitized to any of the species specific (and potentially dangerous) peanut proteins.

Protein stability largely determines allergenicity. Components that are easily denatured by heat or digestive enzymes generally pose less of a risk than components that remain stable. Patients sensitized to these labile proteins are more likely to tolerate or experience only mild, local symptoms to these less stable components, whereas stable protein components are frequently associated with more severe symptoms. In some cases, patients will react to raw foods, but can tolerate the same food if it has been cooked, and the labile proteins denatured. The ingested amount also affects clinical reactivity; sufficiently large amounts of less stable components can also provoke reactions in some patients.

Research has identified the key allergen components primarily responsible for allergic reactions in a number of foods (Table 1). Laboratories now have access to component assays cleared by the U.S. Food and Drug Administration for egg, milk, and peanut with reliable quantitative reporting of specific IgE down to a 0.1 kUA/L LoQ. Component testing can also be performed on the same automated instrument systems as whole-extract allergen assays, which feature walk-away productivity and are classified as having moderate complexity by CLIA.

Allergen components are identified by the first three letters of the genus name, the first letter of the species name, and a number (the numbers are assigned sequentially in the order they are discovered). Peanut is Arachis hypogeae, so peanut components are called Ara h 1, Ara h 2, etc. Cow’s Milk components all carry the Bos d designation, and Hen’s Egg components the Gal d label.

Allergenic proteins are also grouped into families depending on their structure and function. Profilins, PR-10 proteins, lipid transfer proteins (LTPs), and storage proteins are most relevant to plant-food allergies. Profilins are small proteins widely distributed in distantly related species. They play a role in cross-reactivity between foods and pollens and are degraded by heat and enzymes. The PR-10 proteins, such as Ara h 8, are also widely distributed and cross-reactive, heat-labile proteins, primarily found in the pulp of fruits. Sensitization can lead to local reactions. Some sensitized patients exhibit oral allergy syndrome (OAS), a mild to moderate oral contact urticaria associated with these PR-10 proteins. LTPs, including Ara h 9, are small, stable proteins found mainly in fruit and vegetable peels. They are highly resistant to heat and digestive enzymes, such as gastric acid, and can cause systemic reactions in sensitized patients. Sensitized patients may also have some risk of anaphylaxis, even if LTP-containing foods are processed or cooked. Storage proteins, such as Ara h 1, 2, and 3 from the peanut, commonly cause systemic reactions, and sensitization constitutes an important marker for severe symptoms. Like the LTPs, storage proteins in processed or cooked foods can cause reactions.

Foods of animal origin (for example, Cow’s Milk and Hen’s Egg), also contain a variety of potential allergenic proteins of differing characteristics. Casein, which comprises 80% of whole Milk protein, and whey, which comprises the remaining 20%, each contain allergenic proteins.⁴ Components Bos d 4 (α-lactalbumin) and Bos d 5 (β-lactoglobulin) are susceptible to heat denaturation,⁵ whereas Bos d 8 (casein) is heat-resistant.⁶ Patients who are sensitized to Bos d 8, casein, are unlikely to become tolerant to Cow’s Milk or other casein-containing products.

The most relevant allergenic components in Hen’s Egg are Gal d 2 (ovalbumin), which is susceptible to heat denaturation, and Gal d 1 (ovomucoid), which is not.⁷ Children sensitized only to Gal d 2 are likely to outgrow their egg allergy,⁸ but sensitization to Gal d 1 is rarely outgrown.⁹

Applying CRD in clinical practice

Peanut. Let’s return to the teenager mentioned earlier. She has a history of nausea and stomach cramps if she eats “too many” peanuts, but can tolerate a few peanut M&Ms. Her CRD results show mild/moderate sensitization to Ara h 8, and no sensitization to Ara h 1, 2, 3, and 9. Ara h 8 is a PR-10 protein that is highly cross-reactive with pollens¹⁰ and, by itself, is associated with a low risk of systemic reactions.¹¹ Ara h 9 often accompanies sensitization to other peanut proteins,¹² and is cross-reactive with pitted fruits.¹³ Sensitization to the storage proteins Ara h 1, 2, or 3 is often associated with a risk of systemic reactions, including anaphylaxis.¹⁴ The good news for this teenager is that her test results indicate that she may be a good candidate for OFC with peanut.

Contrast her case with a toddler who developed flushing and rhinorrhea immediately after taking a bite of a peanut butter sandwich. Forty minutes later he began vomiting and developed hives. His peanut component test results showed sensitization to Ara h 1, 2 and 3. This pattern of sensitization, in conjunction with his past clinical history of systemic reaction upon exposure to peanut, puts him at risk for a severe allergic reaction. Based on test results and clinical history, a supervised OFC may not be appropriate. Management considerations could include a peanut-free zone established for his safety, notification of his family, colleagues, and teachers of his peanut allergy, and a prescription for an epinephrine auto-injector.

Milk. Cow’s Milk sensitization is common among young children, with the first reaction in those sensitized occurring at a mean age of two years.¹⁵ Cow’s Milk allergy is another situation where just knowing that there is sensitization to the whole allergen does not tell the entire story; it is also helpful to know to which proteins the patient is sensitized. Children who are sensitized to Bos d 4 and/or Bos d 5 alone, while at risk for a reaction to fresh milk, may tolerate milk in baked products. In these situations, when there is no detected IgE to Bos d 8, an oral food challenge may be appropriate. Sensitization to Bos d 8, casein (with or without sensitization to Bos d 4/Bos d 5) is another matter. Patients with this pattern of sensitivity are at risk for an allergic reaction upon exposure to both fresh milk as well as baked products containing milk. In other words, they should avoid all forms of milk, including yogurt, cookies, cakes, and products processed with milk, such as chocolate, sausage and potato chips.¹⁶ Casein-sensitized children will probably not outgrow their milk allergy.

Hen’s Egg. Hen’s Egg sensitization is also common in young children. There are two major allergenic proteins found in egg white; one is heat-stable and the other is heat-labile. Ovalbumin (Gal d 2) is a heat-labile protein, and patients with Gal d 2 sensitization should avoid uncooked eggs; an OFC with well-cooked egg may be an option. Children with ovalbumin sensitivity alone may develop tolerance later in life and can be tested periodically to help determine if tolerance has developed. Children sensitized to ovomucoid, (Gal d 1) on the other hand, may need to avoid all forms of egg, and are unlikely to develop tolerance. 

Component resolved diagnostics in context

Component resolved diagnostics complements traditional whole extract specific IgE testing by providing physicians additional information to better guide their clinical decision making. The awareness and body of evidence supporting the use of CRD has grown significantly over recent years, and the FDA clearance of Peanut Components has helped to push this new technology into the armamentarium of the allergy specialist. Making these tests available through your laboratory will help to meet the growing demand.