Advances in allergy testing

Oct. 19, 2022

Allergies occur when the immune system reacts to typically harmless substances in the environment. The immune response to an allergen begins with a process called sensitization. Sensitization occurs upon first contact with an allergen, when activated allergen-specific T cells produce IL-4 and IL-13, which causes B-cell isotype class switching to immunoglobulin E (IgE) secreting plasma cells. Sensitization is complete upon the production and release of IgE antibodies, which can bind to the specific allergen.1 Once released into circulation, the IgE antibodies bind to mast cell receptors leaving the allergen-specific receptor site open for future interaction with the allergen. Upon re-exposure, the binding of the allergen to IgE causes a signaling process that results in the release of mediators of inflammation such as histamine, prostaglandins, leukotrienes, and thromboxane, which produce local tissue responses characteristic of an allergic reaction.1 In summary, once an individual is sensitized, symptoms can manifest upon the next contact with the allergen leading to serious or even life-threatening complications.

Allergens can be characterized into four categories including inhalants, contactants, ingestants, and injectants.2 Inhalants are allergens that are breathed in such as pollen, mold, dog dander, and bird feathers. Contactants are allergens that come into direct contact with the skin such as dyes, fabrics, or plant toxins. Ingestants most commonly refer to foods such as shellfish, peanuts, milk, or medications that are taken orally. Injectants refer to medications, bug bites, or insect venom that penetrate the skin. Pollen allergies are the most frequent inhalation allergies (particularly birch and grass pollen). Patients with multiple pollen sensitizations exhibit symptoms of rhinitis, conjunctivitis, and allergic asthma.3 Food allergies manifest with symptoms of burning or itching in the oral cavity, nausea, gastrointestinal spasms, diarrhea, and skin rashes. In severe cases, asthma or anaphylaxis may occur. Sensitizations to peanuts, milk and eggs are the most common to occur in childhood.4 With the high risk of severe adverse reactions to foods like peanuts, an accurate diagnosis of such allergies in children is crucial.

In vitro diagnostics for the detection of specific IgE

In vitro diagnostic tests for the detection of allergen-specific IgE in serum complement conventional allergy diagnostic techniques such as the skin-prick test.5 Such in vitro tests offer higher reproducibility and have the advantage of not being influenced by ongoing symptoms or treatment (antihistamines or anti-inflammatory therapy). These immunoassay blood tests are also a safer option preferred by individuals who are at risk of severe allergic reaction if in contact with even a small amount of allergen.

Immunoassays for the detection of specific IgE have enabled the direct and objective measurement of the extent and specificity of the immune response.6 Such assays for the detection of specific IgE may be based on whole extracts of the allergen source or on individual protein components of the allergen. Two types of immunoassays are radioallergosorbent tests (RAST) and enzyme-linked immunosorbent assays (ELISA).

With RAST, a patient’s serum is incubated with allergens on paper discs or polyurethane caps. A colorimetric reaction is measured in response to the binding of specific IgE to the allergen. The results of RAST testing correlate well to the presence of IgE antibody in serum and skin and provocation tests. However, RAST results to a specific allergen demonstrate specific IgE sensitization but are not proof that the allergen is the cause of clinical symptoms.1

ELISA is a direct binding assay using antibodies or antigens that provides a total measurement of IgE in patient serum. Additionally, measurement of total IgE can serve as a screening test to identify if an allergic reaction may occur and aids in the differentiation of allergic and intrinsic asthma, allergic and vasomotor rhinitis, or atopic and seborrheic dermatitis.

Multiparameter immunoblots to identify multiple sensitizations

With immunoblot technology, the detection of allergen-specific IgE can be achieved for a wide range of allergens, all in one test.7 The use of individual membrane chips means that allergens with widely different properties can be combined on one strip. Either whole extracts of the allergen source or defined partial allergens may be used. This technique requires only a small amount of serum volume (around 100 µL) making it ideal for use in pediatrics. Band signal intensities are converted into (EAST) classes from 0-6. The EAST class system is used in allergy diagnostics and is identical to RAST with respect to the concentration grades.

Molecular allergology or component diagnostics

In contrast to traditional allergy diagnostics, which utilize whole allergen extracts, molecular allergology uses defined single allergen components for the detection of specific IgE.8 These components are highly purified proteins either isolated from the allergen source or produced recombinantly. The use of single allergen components allows for a high level of standardization compared to using allergen extracts. With molecular allergology, the exact trigger of the allergy can be identified, which aids in assessing the risk of a patient to severe allergic reaction and helps in determining appropriate therapy.

Different allergen components elicit different symptoms. For example, patients sensitized to allergens from the family of profilins are expected to have mild symptoms while those sensitized to allergens in a family of storage proteins are expected to have life-threatening reactions.8 Therefore, molecular profiling can determine whether a patient has a high or low risk of a severe allergy reaction. Further, different allergens have different heat stability. Therefore, certain heat-labile components in foods (profilins, PR10 proteins) are denatured by cooking processes, which reduces the risk of reaction. Because of this, molecular allergology can be used to determine the level of risk a patient has to a certain allergen and whether that risk could be lessened by cooking processes.

Extract-based allergen assays are a screening tool for IgE antibodies but suffer from issues with cross reactivity. Cross reactions arise due to structural similarities among proteins from different sources. This problem can be resolved using purified single allergens in molecular allergology, which allows for the discrimination of primary sensitizations from cross reactions. Further, identifying the specific allergenic component to which the patient has been sensitized can guide recommendations for avoidance of likely cross-reactive triggers.

Pinpointing the precise trigger of the allergy is also critical to determining proper therapy. However, traditional skin prick tests and extract-based allergen assays commonly show that a patient has reactions to multiple allergens, making treatment decisions difficult. While this may be a true polysensitization, it is also possible for the patient to have a monosensitization with cross reactions. About 50–60% of all skin prick tests yield false positive results.9 Allergies can be treated by either avoidance of the allergen or with specific immunotherapy (SIT).10 As SIT is most successful when a patient is primarily sensitized to the major component of the allergen extract, it is important to identify the exact allergen trigger. This way, ineffective SIT can be avoided.

Cross reactive carbohydrate determinants

Many allergens are glycoproteins that contain oligosaccharide side chains. These carbohydrate side chains can complicate in vitro diagnostics as some patient samples contain specific IgE antibodies against these structures. Reactions with these so-called cross-reactive carbohydrate determinants (CCDs) do not have a clinical relevance, so it is important to consider whether a positive test might be due to the presence of anti-CCD antibodies.7 Allergy tests that provide a CCD control allow you to evaluate whether anti-CCD IgE antibodies are present and possibly leading to a positive test result. Reagents to remove anti-CCD IgE are available so that analysis can be repeated without these nonspecific reactions. Anti-CCD reactions only occur with assays based on allergen extracts and native components, as recombinantly produced protein components do not undergo posttranslational glycosylation.

Comprehensive and targeted profiles

Whole allergen extracts are available for a wide range of allergen sources. With immunoblot multiparameter testing, different profiles can be created for specific applications such as food, inhalation, or atopic testing as well as country- and region- specific profiles.7 For example, profiles can be selected that focus on the food or inhalation allergens of particular relevance to the Middle East region, the Gulf states, the Mahgreb, Iran, Lebanon, the Mediterranean region, Turkey, India, China, or Southeast Asia.

For certain applications, extracts and defined partial allergens may be combined into one profile. This allows for the simultaneous investigation of sensitizations to different whole allergens and identification of important allergic reactions in one test.

At-home allergy testing

With direct-to-consumer testing, it is possible to do an allergy test from the comfort of your own home. With a finger prick using a sterile lancing device, drops of blood are added to a special filter paper called a dried-blood spot (DBS) card, which can then be sent to a laboratory for analysis. Following the laboratory testing, results are shared with a physician. Finally, the physician discusses these test results with the patient. DBS testing offers the advantage of being less invasive and more convenient as the sample can be self-collected at home by patients.


The availability of well standardized in vitro allergy tests has greatly improved the quality of allergy diagnosis. The development of multiparameter immunoblot tests has allowed for the determination of multiple sensitizations in one test. Further, molecular allergology has enabled the identification of the particular allergy triggering component, which is important in assessing a patient’s risk of severe allergic reaction. Inclusion of CCD controls helps in the differentiation between cross reactions and multiple sensitizations. Such developments are necessary for optimizing patient management and for selection of the appropriate immunotherapy.


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