Molecular Allergy Diagnostics
المؤلف:
Marcello Ciaccio
المصدر:
Clinical and Laboratory Medicine Textbook 2021
الجزء والصفحة:
p505-506
2025-12-16
59
Starting from the mid-nineties of the last century, in parallel with the development of proteomic techniques, we have witnessed the birth of molecular allergology, which has provided various confirmations and numerous new findings in the field of allergology. In particular, the following must be mentioned:
• Most allergens possess high antigenic complexity.
• The individual response to an allergen depends on the genetic background of each subject. Several allergens can present structural homology, which can determine phenomena of cross-reactivity due to the recognition of different allergens by the same antibody.
• IgE generally tends to recognize conformational and non linear epitopes. Indeed, in the case of food allergies, only molecules that maintain their three-dimensional structure even at high temperatures can induce allergy in sensitized patients.
The availability of recombinant allergens and/or highly purified extracts has allowed the introduction of molecular diagnostics (Component Resolved Diagnosis, CRD), which allows the analysis of IgE reactivity to individual allergens.
CRD allows the characterization of the specific allergological profile of an individual, allowing the distinction between primary or genuine sensitizations and cross- reactivities. The CRD has, therefore, revolutionized the clinical management of the patient allowing, for example, the selection of patients who can benefit from specific immunotherapy (ITS), or the assessment of the severity of the reaction to food allergens, allowing to distinguish between subjects sensitive to highly stable molecules and subjects sensitive to thermal and gastro-labile molecules, which will develop only oral allergy syndrome (OAS) after ingestion of raw food.
Analytical Methods for Molecular Diagnostics
CRD can be performed through the use of:
• Single molecular components (singleplex), which allow a targeted diagnosis
• Matrices, which consist of many allergens deposited on microarrays (multiplex)
Singleplex Molecular Diagnostics
Monoplex molecular diagnostics consists of a second-level test to confirm a diagnostic suspicion, allowing the identification of specific IgE toward a specific allergen. It has the advantage of being performed on the same analytical plat form (quantitative, highly automated) where specific IgE toward extractive allergens are sought; this allows the use of algorithms by a reflex test approach so that the execution of few and targeted analytical assays determines an appropriate and efficient use of the available economic resources. In this approach, however, there is a risk of underestimating the presence of other unsuspected sensitizations by identifying only the components hypothesized a priori. The principle of the method is the same as the immunoassays described above for Cap FEIA and 3gAllergy consolidated on the same instruments used for the research of specific IgE, except allergens that in the determination of allergenic molecules will be recombinant or native allergens.
Multiplex Molecular Diagnostics (Microarrays)
Microarray diagnostics is a third-level test that allows the definition of the allergy profile of a patient. Currently, a microarray (ImmunoCAP ISAC, Thermo Fisher Scientific, Waltham, MA, USA) is available on the market that allows the simultaneous determination of IgE directed toward more than a hundred different allergens using a small amount of serum (30 μL). However, interpreting this test is somewhat complex and requires highly specialized personnel.
The test is also semi-quantitative, calibrated against an internal standard, and has lower diagnostic accuracy than the Cap FEIA test.
Table 1 describes the main characteristics of the two diagnostic systems. From the point of view of purely productive efficiency for the resources used, diagnostics using the microarray, based on the current costs envisaged for carrying out the tests, is advantageous if the patient’s clinical situation requires the search for specific IgE toward several molecular components greater than 12–13 allergens.
Table1. Advantages and disadvantages of ISAC multiplexes and Cap FEIA singleplex
Microarray Diagnostics (ImmunoCAP® ISAC).
Fluorescent antihuman IgE antibodies are used to detect antigen–antibody binding between specific IgE present in the patient’s serum and antigens conjugated to a solid phase on a slide (chip). The fluorescence is subsequently measured by a scanner equipped with a laser excitation source. A densitometry software then analyzes the image and provides the test results as a function of the fluorescence intensity detected on each spot.
The test takes approximately 5 h to perform. The results are processed as ISAC classes (absent-low-medium-high) and international system units, providing a semi-quantitative IgE determination based on a specific reference curve. The system has high diagnostic reliability as each molecule is tested in triplicate.
Microarray-based diagnostics could be particularly useful in the diagnostic framing of complex clinical situations, such as:
• Patients with multiple sensitizations, not identifiable based on history and first-level tests
• Patients who do not respond to ITS therapy for whom it is appropriate to evaluate the presence of other possible allergens
• Patients with “idiopathic” anaphylaxis, to identify any sensitization not diagnosed by traditional tests
• Pediatric patients for whom it is not possible to obtain a sufficient sample to perform the analysis by monoplex diagnostics
However, there are some limitations to the use of multiplex technology:
1. The test is not automated and, therefore, is rather com plex to perform and subject to a higher risk of errors.
2. Reduced analytical sensitivity and specificity characterize it compared to the single-plex method and a high rate of false positives.
3. The interpretation of the results is complex and must be performed by a specialist with a good knowledge of the various molecules and their diagnostic significance.
A new test system (ALEX2, MacroArrayDX, Wien, Austria) has recently been introduced for simultaneous detection of Total IgE and Specific IgE to 117 extracts and 183 molecules by solid-phase enzyme immunoassay. Extract and allergen molecules combined with nano-particles are sorbed on a solid-phase substrate, forming a macroscopic multiplex matrix - the immune allergy chip.
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