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2022 NOT OPEN

Platteel; A comprehensive comparison between ISAC and ALEX2 multiplex test systems

The aim of the present study was to analyze the performance of two different multiplex allergy test systems, ImmunoCAP ISAC® and ALEX2® Allergy Explorer using serum samples of 49 patients preselected based on positive ImmunoCAP ISAC® results. A good overall agreement (94%) and positive agreement percentage values between ISAC and ALEX2® has been observed. 59% of the qualitative discrepancies were related to weak positive results. Remarkably, the overall agreement of dichotomous results (negative, positive) was found to be identical (94.3%). However, the CCD inhibition step to block CCD-specific IgE antibodies used in the ALEX2® test revealed to be innovative and essential to obtain higher clinical specificity. Anti-CCD antibodies are reported up to 30%. Interestingly, the patient with the highest reactivity against MUXF3 (CCD) on ISAC (8.5 ISU) revealed, besides two double positive results for wasp venom (Pol d 5 and Ves v 5), 7 ISAC+/ALEX− results all of which were native proteins of plant origin and/or plant food related allergens (Cry j 1, Cup a 1, Cyn d 1, Gly m 6, Jug r 2, Sal k 1, Tri a aA_TI). Here false positive results were generated using the ISAC test.

ALEX2® was able to score plus points with the following aspects:

  • measurement of total IgE

  • absorption of sIgE against CCDs

  • more comprehensive allergen panel

  • inclusion of minor allergen components enhances clinical value

doi: 10.1515/cclm-2022-0191

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2022 NOT OPEN

López-Rodríguez; Serodominance Profile in a Dust Mite Complex Region

To investigate the molecular sensitisation profile of mite allergic patients in a region with high environmental house dust and storage mite occurrence, a specific molecular allergen panel using the Allergy Explorer ALEX2 was correlated with clinical parameters.

All subjects with a positive skin prick test exhibited a positive response to one of the tested molecular allergens. 66% of the cohort recognised Der p 1, 78% Der p 2 and 86% Der p 23. The great advantage of the ALEX2 test was the additional evaluation of IgE responses to mid-tier and minor house dust mite allergens. Der p 5 revealed IgE-reactivity in 44%, Der p 7 in 36%, Der p 20 in 10% and Der p 21 in 40%. Der p 7 levels were higher among rhinitis patients. In contrast, the atopic dermatitis relative risk was increased in patients sensitised to Der f 2, Der p 2, and Der p 23. Sensitisation to Der p 10 decreases the risk to have atopic dermatitis.

The use of multiple molecular panels allows the improvement of patient management and to evaluate the risk for atopic dermatitis or other conditions like asthma or rhinitis.

doi: 10.1159/000523869

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2022 OPEN ACCESS

González-Pérez; Storage Mite Precision Allergy Molecular Diagnosis in the Moderate-to-Severe T2-High Asthma Phenotype

In the present study, storage mite sensitisation profiles were determined in two different (moderate and severe) asthma phenotypes.

Methodically, IgE Western Blotting, determination of blood eosinophils as well as serological analysis using the Allergy Explorer ALEX2 were performed.

Mite group 2 allergens were most frequently recognised. Lep d 2 (83%) was the most recognised, followed by Gly d 2 (69%) and Tyr p 2 (47%) in 133/164 asthmatic patients. The majority of storage mite sensitised individuals (99%) were simultaneously sensitised to house dust mites. In respect of group 2 allergen interactions, above 90% showed a sIgE response to Der p 2.

Actually, Lep d 2 and Gly d 2 may be speculated as potential biomarkers in milder forms of asthma presentations. However, storage mite sensitisation demands specific attention, as current diagnostic and therapeutic tools are based on Dermatophagoides allergens. Individual sensitisation patterns need to be carefully evaluated for precise diagnosis and targeted allergen-specific immunotherapy.

doi:10.3390/ijms23084297

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2022 OPEN ACCESS

Grilo; Tropomyosin is no accurate marker allergen for diagnosis of shrimp allergy in Central Europe

So far, shrimp allergy has been considered to result from IgE-cross-reactivity following sensitisation to Der p 10, the tropomyosin from Dermatophagoides pteronissimus. Because tropomyosin-specific IgE was found to be a good predictor of shrimp allergy in HDM-allergic individuals, singleplex assays with shrimp tropomyosin found their way into serological routine diagnosis.

However, evidence accumulated that additional allergens cause shrimp allergy and cross-reactivity with house dust mites, such as arginine kinase (Pen m 2), myosin light chain (Pen m 3), sarcoplasmic calcium-binding protein (Pen m 4), hemocyanin, triose- phosphatase-isomerase (TPI), and troponin C (Cra c 6).

In the present study, 79 individuals who experienced allergic reactions to shrimps and displayed shrimp-specific IgE in ImmunoCAP® were analysed with the Allergy Explorer ALEX2® and immunoblotting.

The ALEX2® test revealed that 42% of the patients displayed IgE to Pen m 1 (tropomyosin, 19% exclusively), 20% to Pen m 2 (arginine kinase, 3.7% exclusively), 10% to Pen m 3 (myosin light chain, 2.5% exclusively), and 11% to Cra c 6 (troponin C).

Thus, tropomyosin represented no major allergen in this cohort. Notably, 30% of the patients recognised Pen m 4 and 16% showed exclusive IgE-reactivity with confirmed clinical relevance. Therefore, Pen m 4 was considered as important as Pen m 1 for the diagnosis of shrimp allergy in Central Europe.

All recombinant allergens together achieved a sensitivity of 68%. It is expected that the inclusion of hemocyanin and ubiquitin might enhance this value as 10% and 9% of the patients recognised the corresponding proteins of 70 and 5–7 kDa, respectively, in the immunoblot.

doi: 10.1111/all.15290

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2022 OPEN ACCESS

Rodinkova; Molecular Profile Sensitization to House Dust Mites as an Important Aspect for Predicting the Efficiency of Allergen Immunotherapy

In a huge patient cohort of more than 10,000 patients from Ukraine, the molecular sensitisation profile to house dust mites was determined with ALEX2 Allergy Explorer. Sensitisation to at least one HDM allergen has been established in 2875 people (27%). The number of sensitised children was 2.26 times higher than that of adults: 69% children vs 31% adults.

Patients were grouped by their sensitisations. Sensitisations to group 2 allergens (Der f 2 – 73%, Der p 2 – 72%) were the most common among the entire group, followed by polysensitisation to group 1 (Der p 1 – 55%, Der f 1 – 54%), group 2 (Der p 2, Der f 2) allergens and Der p 23 (56%). Sensitisations to Der p 21 was found in 27%, to Der p 5 in 28% and to Der p 7 in 23% of the patients. Lowest sensitisation levels were registered for: Der p 20 (8%), Der p 10 (6%) and Der p 11 (0.6%).

To conclude, the key sensitising HDM allergens in Ukraine are Der p 2 and Der f 2. The second most prevalent among adults is monosensitisation to Der p 23, and among children polysensitisation to group 1, group 2 allergens and Der p 23.

doi: 10.3389/fimmu.2022.848616

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2022 OPEN ACCESS

Feuerecker; One Year in the Extreme Isolation of Antarctica—Is This Enough to Modulate an “Allergic” Sensitization?

In this retrospective study, blood samples from people who spent a year wintering in Antarctica and reported increased or new allergic reactions to environmental allergens after their return, were analysed.

Due to the low sample volumes, the objective was to obtain the maximal possible scientific outcome achievable on a retrospective basis. Therefore, ALEX® Allergy Explorer was chosen as it contains a very broad variety of allergen extracts and molecular allergens.

Specific IgE measurements corresponded well to the general allergen situation in various locations. Thus, where there was no pollen exposure, the kinetics also showed a reduction in specific IgE. Five participants showed the highest specific IgE levels after returning to the "normal" world. For house dust and storage mite specific IgE, the examination revealed different kinetics. Six out of 10 had the highest specific IgE concentrations at the inner Antarctic measurement time point.

To conclude, long-term residence in Antarctica can reduce sensitisation to absent allergen sources.

doi: 10.3390/biomedicines10020448

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2021 OPEN ACCESS

Gonzáles-Pérez; House Dust Mite Precision Allergy Molecular Diagnosis (PAMD@) in the Th2-prone Atopic Dermatitis Endotype

Precision medicine including personalised diagnostic and therapeutic strategies requires the depiction of the disease-eliciting allergens by demonstrating the presence of allergen-specific IgE antibodies.

In order to implement precision medicine in patients with atopic dermatitis, the present study investigated the IgE molecular profile to D.pteronissinus in patients with clinically relevant house dust mite sensitisation. Skin prick tests with mite extracts and a panel of 9 individual molecular house dust mite allergens (using ALEX2® Allergy Explorer) were used to perform atopic dermatitis endotyping. 86% of patients presented IgE reactivity to major allergens (Der p 23, Der p 2, Der p 1) and 65% to mid-tier allergens (Der p 5, Der p 7, Der p 21). Der p 23 was the most prevalent allergen recognised in this population.

To conclude, the available house dust mite molecular panel precisely identifies the large majority (>97%) of D.pteronissinus allergic subjects suffering from type-2 atopic dermatitis.

doi: 10.3390/life11121418

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2021 NOT OPEN

Quan P; Validation of the ALEX microarray platform for specific IgE detection of respiratory and plant-food allergens

In this study setup, three different allergy test systems - the Allergy Explorer ALEX2®, ISAC112® and ImmunoCAP® were compared according their technical performance and diagnostic capacity for the diagnosis of various allergies.

ALEX2® showed improved diagnostic capacity for Alternaria and apple allergy compared with ISAC112®, due to the better performance of Alt a 1 and the inclusion of Mal d 3, respectively. It is important to consider that in terms of CRD, extensively wide panels of allergens allow the inclusion of clinically relevant allergens such as Der p 23, absent in ISAC112®.

ALEX2® performed well in technical validation assays by measuring repeatability and inter-assay, inter-batch and inter-lab reproducibility.

doi: 10.1016/j.anai.2021.11.019

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2021 OPEN ACCESS

Scala E; A qualitative and quantitative comparison of IgE antibody profiles with two multiplex platforms for component-resolved diagnostics in allergic patients

In the present study, a cohort of 140 allergic individuals presenting distinct clinical phenotypes (e.g., atopic dermatitis, adverse food reactions, allergic rhinitis, bronchial asthma) were analysed by two different in-vitro diagnostic allergy tests - Allergy Explorer-ALEX2® and ImmunoCAP ISAC112®. A significant conformity was observed when dichotomised data (94.3% concordant results) were analysed. Especially for species-specific marker allergens - perfect correlations were detected. The Allergy Explorer ALEX2® test includes a larger number of allergens, allowing a broader molecular detection. Furthermore, the presence of several allergens only on ALEX2® revealed as an important feature. However, heterogeneous results emerged comparing panallergens.

doi: 10.1111/cea.14016

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2021 OPEN ACCESS

Čelakovská; Analysis of Results of Specific IgE in 100 Atopic Dermatitis Patients with the Use of Multiplex Examination ALEX2—Allergy Explorer

In this study, specific IgE antibodies of 100 patients with atopic dermatitis to the whole ALEX panel were evaluated. Patients with severe AD, showed high levels of specific IgE to proteins of the NPC2 family (storage mites), lipocalin (dog), arginine kinase (moulds, cockroach, house dust mite, shrimp), uteroglobin (cat, rabbit), Mn superoxide dismutase (moulds), PR-10 protein (beech, apple, hazelnut), peritrophin-like domain (house dust mite) and rye pollen. In a subgroup of patients with bronchial asthma, high IgE values against molecular components from house dust mites, storage mites, grass pollen and moulds were observed.

doi: 10.3390/ijms22105286

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2021 OPEN ACCESS

Čelakovská; Atopic Dermatitis and Sensitisation to Molecular Components of Alternaria, Cladosporium, Penicillium, Aspergillus, and Malassezia-Results of Allergy Explorer ALEX 2

Molecular diagnostic tests improve laboratory diagnostics of IgE-mediated allergies. Here, ALEX2- Allergy Explorer was used to determine the sensitisation profile towards moulds and yeast in patients suffering from atopic dermatitis. The relation between the sensitization to molecular components of moulds and yeast and the severity of atopic dermatitis, and the occurrence of bronchial asthma and allergic rhinitis was evaluated. Their results demonstrated that the sensitization to Mala s 6, Mala s 11, Sac c, Asp f 6, Cla h and Cla h 8 correlates to the severity of atopic dermatitis. The sensitization to Sac c, Alt a 6, Cla h, Cla h 8 was observed significantly more frequently in patients suffering from bronchial asthma to Mala s 6 in patients suffering from allergic rhinitis. In patients with severe form of atopic dermatitis (AD), a very high level of specific IgE was recorded to Mala s 11 (in 36%) and to Asp f 6 (in 12%)

doi: 10.3390/jof7030183

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2021 OPEN ACCESS

Villalta; Evaluation and predictive value of IgE responses toward a comprehensive panel of house dust mite allergens using a new multiplex assay: a real-life experience on an Italian population

Specific IgE reactivity to a comprehensive panel of house dust mite allergens such as Der f 1, Der p 1, Der f 2, Der p 2, Der p 5, Der p 7, Der p 10, Der p 11, Der p 20, Der p 21 and Der p 23 was tested in patients with respiratory symptoms using Allergy Explorer (ALEX2®). Prevalence and IgE levels of Der f 1, Der f 2, Der p 1 and Der p 20 were significantly higher in asthmatic patients, whereas subjects negative for minor allergens resulted more frequently suffering from rhinitis. Asthmatic patients had IgE reactivity to a larger number of HDM allergens than patients with only rhinitis.

doi: 10.23822/EurAnnACI.1764-1489.195

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2021 NOT OPEN

Hoang; Modeling the conversion between specific IgE test platforms for nut allergens in children and adolescents

Plasma from Canadian and Austrian children/adolescents with peanut/tree nut sensitisation and a cohort of sensitised, high‐risk, pre‐school asthmatics were measured with three multiplex IgE test platforms: ALEX®, MeDALL‐chip (Mechanisms of Development of Allergy) (Thermo Fisher), and EUROLINE (EUROIMMUN). Using two regression methods, they demonstrated the ability to model allergen‐specific relationships for peanut and tree nut sIgE testing at the extract and molecular‐level, in order from highest to lowest: Ara h 2, Ara h 6, Jug r 1, Ana o 3, Ara h 1, Jug r 2, and Cor a 9.

doi: 10.1111/all.14529

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2021 OPEN ACCESS

Pinheiro; En route to personalized medicine: uncovering distinct IgE reactivity pattern to house dust mite components in Brazilian and Austrian allergic patients

A new in vitro multiplex allergen assay (ALEX) which allows simultaneous measurement of specific IgE against the whole allergen extract as well as its components were used. The data showed that in Brazil the inclusion of the molecular components Blo t 5 and/or Blo t 21 major allergens and Blo t 2 can increase the sensitivity and specificity of the assay for the diagnosis of allergy to B. tropicalis. Furthermore, monosensitisation to Blo t 2 was found in some individuals. Regarding the sensitization profile of individuals sensitized to D. pteronyssinus, they demonstrated that molecular components Der p23 and Der p 7, in addition to Der p 1 and Der p 2 are essential for an accurate diagnosis.

doi.org/10.1002/clt2.12004

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2020 NOT OPEN

Gonzáles-Pérez; Mite molecular profile in the Th2-polarized moderate-to-severe persistent asthma endotype subjected to high allergen exposure

González-Pérez et al. confirmed the dominant role of sIgE sensitization to Der p 2 and Der p 23 (86%) and Der p 1 (74%) among an asthmatic population in Spain with moderate-to-severe symptoms. Their results also demonstrated that more than 70% of the patients were sensitized to any of the major allergens, however an inclusion of allergens such as Der p 5, Der p 7 and Der p 21 enhanced diagnosis of mite-sensitized asthmatics up to >94%.

doi: 10.1159/000510118

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2020 OPEN ACCESS

Koch; IgE multiplex testing in house dust mite allergy is utile, and sensitivity is comparable to extract‐based singleplex testing

Another study compared allergy tests such as singleplex assay ImmunoCAP® (detecting Der p 1, 2, 10 and 23), the multiplex assay ImmunoCAP® ISAC (detecting Der p 1, 2 and 10) and Allergy Explorer (ALEX®) versions 1 and 2 (detecting Der p 1, 2, 5, 7, 10, 11, 20, 21 and 23). In general, sensitivity of molecular tests was lower compared to singleplex extract-based testing. However, sensitivity increased the more HDM allergen molecules were included in the test. Therefore, sensitivity of ALEX2® was higher compared to all other molecular assays. A clear correlation according to sensitivity between molecular test systems with HDM sIgE levels was observed implicating that higher sIgE levels lead to better sensitivity of molecular testing. Der p 1, 2 and 23 were confirmed to be major allergens. Moreover, Der p 23 is the third major HDM allergen and therefore essential to include in molecular test systems. Importantly, ALEX2® performed statistically equal to extract-based diagnosis in patients with allergen-specific IgE levels higher that 1.0 kU/L with a sensitivity of 97.2%.

doi: 10.1111/all.14271

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2020 OPEN ACCESS

Gellrich; De novo sensitization during subcutaneous allergen specifc immunotherapy - an analysis of 51 cases of SCIT and 33 symptomatically treated controls

To study the risk of subcutaneous allergen specific immunotherapy (SCIT)-induced neosensitisations, total IgE, sIgE reactivity and sIgG patterns to purifed natural allergen extract and allergen components were retrospectively measured using ALEX®. In 24% of SCIT-treated patients, they found new sIgE against allergen components of the allergen source treated by SCIT. In the SCIT-treated group, neosensitisation affected major and minor allergen components, and was accompanied by a strong induction of sIgG against major components. Patients undergoing SCIT might carry an enhanced risk of neosensitisation towards formerly unrecognized allergen components. According to anamnestic data, these neosensitisations might be of clinical relevance - supporting attempts towards personalized recombinant vaccines.

doi: 10.1038/s41598-020-63087-4

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2020 OPEN ACCESS

Rauber; The honey bee venom allergen Api m 10 displays one major IgE epitope, Api m 10160‐174

Nowadays, honey bee venom (HBV) allergy is treated by allergen immunotherapy (AIT) whose efficacy is low. Api m 10 was identified as a major allergen in HBV allergy, however the quantity of Api m 10 in the venom is of low abundance (approx. 1%). This may explain a potential link between underrepresentation in several therapeutic HBV preparations and treatment failure in dominantly Api m 10 sensitised HBV-allergic patients. In theory, this lack couled be compensated by spiking therapeutic HBV preparations with rApi m 10. However, Api m 10 is easily degraded. Therefore, alternative strategies need to be developed. To characterize the IgE response to Api m 10 in detail, linear IgE epitopes recognized by patients allergic to HBV were investigated. Synthetic peptides spanning the whole amino acid sequence of Api m 10 were coupled to macroarrays (Macro Array Diagnostics). Interestingly, one peptide, P54 (Api m 10160‐174; amino acid sequence: ADSDVTTLPTLIGKN), was recognized by 100% of the Api m 10‐positive sera and hence represents the dominant linear IgE epitope of Api m 10. A short immunodominant peptide is easier to produce, can be included in therapeutic preparations and thus may have relevant advantages over the use of full‐length rApi m 10.

doi: 10.1111/all.14187

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2020 OPEN ACCESS

Kowalczyk; Anaphylaxis in an 8-Year-Old Boy Following the Consumption of Poppy Seed

This case study describes a young patient with poppy seed allergy. Hazelnut allergy was found to co-occur in this patient. However, the primary responsible allergen was considered to be poppy seed.

doi: 10.18176/jiaci.0493

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2019 NOT OPEN

Scala; β‐1,3‐glucanase rOle e 9 and MnSOD rAsp f 6 IgE reactivity are the signature of atopic dermatitis in the Mediterranean area

To identify patterns of IgE sensitisation in patients with atopic dermatitis, ImmunoCAP ISAC® and Allergy Explorer‐ALEX® microarray analysis were performed. The olive tree pollen β‐1,3‐glucanase rOle e 9 and the manganese superoxide dismutase from Aspergillus rAsp f 6 were the molecules most significantly associated with AD occurrence and allowed to discriminate among the moderate and severe forms of disease. This publication demonstrates the importance of allergy tests using a large panel of molecular components to unveils important associations between IgE reactivity profiles and AD clinical presentation.

doi: 10.1111/cea.13555

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2019 NOT OPEN

Buzzulini; Evaluation of a new multiplex assay for allergy diagnosis

In a selected population of 105 allergic patients, skin prick test (SPT), singleplex (ImmunoCAP®) and multiplex assays (Allergy Explerer ALEX®) were compared. IgE antibodies to pollen, dust mites and food allergens were detected. Their results demonstrated a substantial agreement between ALEX® and SPT. When IgE to extracts was detected, ALEX® and ImmunoCAP® showed comparable results for inhalant and food allergens. In addition, using molecular components a higher agreement was observed for inhalant and food allergen molecules. Finally, the authors appreciated that ALEX® comprises all advantages of a multiplex test, such as an easy to learn method and a precise identification of the disease-eliciting molecules. To their opinion, ALEX® gives a boost to research in Molecular Allergy.

doi: 10.1016/j.cca.2019.02.025

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2019 NOT OPEN

Hoang; Extract and component-specific sensitization patterns in Canadian moderate-to-severe preschool asthmatics

In a cohort of North American preschool asthmatics, peanut and animal allergens were identified as major allergen sources. The dataset of this study provides detailed information on molecular diagnosis in preschool asthma. In particular, seed storage proteins (Ara h 1, 2, 3, 6), uteroglobin from cat (Fel d 1), and lipocalin from dog (Can f 1) demonstrated the strongest linkage to clinical markers of asthma severity. Allergen molecules can be biomarkers for stratification and endotyping in future prospective trials.

doi: 10.1111/all.13927

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2019 OPEN ACCESS

Bojcukova; Comparison of two multiplex arrays in the diagnostics of allergy

Based on Heffler et al., the present study went a step further and performed a comparison between the results of ALEX® and ImmunoCAP® ISAC for specific allergen components (most common allergens in these patients) such as: IgE against tree pollen Bet v 1, grass pollen Phl p 1 and 5, house dust mite Der p 1 and 2 and cat Fel d 1. They concluded a good correlation between ALEX® and ISAC.

doi.org/10.1186/s13601-019-0270-y

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2018 OPEN ACCESS

Heffler; Extended IgE profile based on an allergen macroarray: a novel tool for precision medicine in allergy diagnosis;

A very interesting study of 43 patients with allergies comparing two multiplex test systems (ImmunoCAP® ISAC and ALEX®) based on allergen extracts and molecules, found a good correlation between these methods. Several advantages of ALEX® were monitored, such as the ability of measuring total IgE, the high number of 282 reagents (157 extractive allergens and 125 molecular components) compared to 112 different components (both extracts and molecular allergens) in ISAC as well as the inhibition of CCD (Cross-reactive Carbohydrate Determinants) reactivity further improves the specificity of the IgE assay.

doi: 10.1186/s40413-018-0186-3

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