Evaluation of major mite allergens from European standardized commercial extracts for in vivo diagnosis: addressing the need for precision medicine

Allergen standardization sets the basis for better and more reproducible allergen extracts. Regulatory agencies provide evidences—Good Manufacturing Practices—for the manufacturing and quality control of licensed allergenic extracts concerning the need for product specifications, analytical procedures, process validations, microbiological attributes, and labelling [25‐27]. However, most existing regulations exempt non-standardized or “named-patient products” from the more rigorous licensing requirements in terms of safety and efficacy [28].

The variations in the biological activity among allergen extracts and manufacturers are dependent on the origin of the source material, the extraction and reconstitution process—based on arbitrary in-house reference standards—and also the clinical features of the referenced allergic subjects [29, 30]. Currently, mites are being commercially grown on a variety of food substrates, and the purity of the mite bodies in the total extract varies among the different sources and different countries [31]. In fact, it has been proven that mite-culturing diet directly effects population growth, the dynamics of allergen accumulation, the group 1/2 allergen ratio and the endotoxin contents in extracts of cultured HDM [32]. The results of our study indicate that certain natural Der p. and B. tropicalis extracts lack important allergens showing a considerable variability in the allergen composition and content. The protein content and the major allergens—Der p 1, Der p 2, and Blo t 5—found in the individual extracts was quite heterogeneous among the different manufacturers. The Der p. extracts showed a tenfold variation of the total protein content and the B. tropicalis extracts an almost 60-fold variation. The levels of the major allergens of Der p., Der p 1, Der p 2, ranged from 1.21 to 30.6 μg/mL (a 30-fold variation) and 0.55–20.49 μg/mL (37-fold variation) respectively, showing a quantitative lower range compared to those commercial extracts previously published in Italy [33] (Der p 1: 9.6–36.2 μg/mL, Der p 2: 0.7–31.7 μg/mL), Austria [12] (Der p 1: 6.0–40.8 μg/mL and Der p 2: 1.7–45 μg/mL) and Korea [34].

In addition, the Der p 1/Der p 2 ratios differed considerably among extracts from 0.28 to 15.62 showing lower levels compared to previous reports from different European and Asian manufacturers [35]. Interestingly, the concentration of Der p 1 in the individual extracts was greater than Der p 2, except for extracts #1 and #4. This variation in ratios might be related to the source material—mite feces and/or of mite bodies-, used for extraction from each manufacturer [36].

Although more than 23 D. pteronyssinus allergens have been characterized [37], several studies have indicated that group 1 and 2 allergens represent the clinically most important mite allergens, which are in fact the major allergens in our population [38, 39]. It has been shown that a combination of both D. pteronyssinus. Allergens allow diagnosis of more than 95% of patients with HDM allergy [40]. In contrast, Casset et al. [12] confirmed no correlation between Der p 1 or Der p 2 concentrations in the extracts and the intensity of the skin response, indicating the relevance of minor allergens such as Der p 5, 7, 10 and 21 for the biological response.

In the present study, the major allergen from B. tropicalis Blo t 5 ranged from 0.0 to 2.15 μg/mL. It is remarkable that Blo t 5 was only detected in 3 out of the 7 commercially available extracts—a 53-fold variation—from B. tropicalis. Interestingly, relevant components belonging to Blot4 (56 kDa)—a locally serodominant regional allergen in China [41] and Spain [42]—were only detected in the B. tropicalis extracts from manufacturers 1 and 5, therefore a precise diagnosis may not be achieved with all the tested reagents in specific populations.

In this regard, Cardona et al. described the kinetics of released allergens during the growth cycle of B. tropicalis and B. kulagini confirming that mite cultures during the maximum growth contain the largest number of allergenic components as well as the highest Blo t 5 concentration [43].

Since both the protein and the major allergens contents may not necessarily have an impact at the appropriateness of the extracts for diagnosis, in terms of their potential IgE binding, CAP inhibition assays with 16 extracts of Der p. and B. tropicalis and a pool of sera of moderate-severe rhinitic subjects were carried out to confirm variable patterns of IgE recognition—specially for B. tropicalis—depending on the evaluated extract (Fig. 2).

The present results are similar to previous studies that pointed out marked differences from different manufacturers confirming that SPT extracts are so heterogeneous that a few of them are incapable of showing a positive response in some patients, stating that absence of important components can affect the diagnosis of HDMs allergy [33, 44, 45]. The current data display that no clear-cut progress have been made yet concerning this issue.

Although the commercial extracts for Der p. and B. tropicalis included in the study claimed to be biologically standardized to each in-house reference standards, wide differences in terms of allergen composition and content were found. Despite SPT has been previously described as reproducible [7], the results of this study are in not in line with this remark regarding the accuracy of HDM allergy diagnosis. However, it has been shown that standardized extracts are not necessarily more potent than conventional extracts based on weight per volume (w/v) or protein nitrogen units [46].

Mass spectrometry (MS) has been used as a tool to identify novel allergens or to prove identity and enhance the quality control of recombinant allergens [47]. Spiric et al. [48] suggest that a MS technique known as multiple reaction-monitoring (MRM) may complement the overall potency measurements or quantitation of allergenic proteins by ELISA to inform practitioners of compositional differences among HDM reagents, and of variations in composition among lots from each manufacturer.

Our investigation has some limitations, the possibility of degradation resulting from storage condition and duration of storage may be responsible for some of the variation among extracts, even though the reagents were properly stored and evaluated within the documented expiration date. Noteworthy, Jeong et al. [49] showed that despite almost 40% of total protein was degraded, more than 90% of IgE reactivity was preserved during the first 2 months of storage when reconstituted in 50% glycerol and about 80% of Der p 1 content was still preserved in the extracts. Although quantification of relevant allergens for Der p. (Der p 5, 7, 10, 21 and 23) and B. tropicalis (Blo t 4 and 21) was not performed, stain intensities for specific components were variable or even absent among extracts from different manufacturers in a few Der p. (extracts #4, 5, 6 and 9) and B. tropicalis (extracts #2, 3, 4, 6 and 7).

The probability of a given sensitization to be clinically relevant depends on the type of allergen and country where the patient lives [50]. The findings of the current study provide relevant information for the daily allergy practice in search of a more accurate diagnosis. Although, variation in therapeutic extracts [51] have been elegantly demonstrated before, no studies comparing commercially available D. pteronyssinus and B. tropicalis SPT reagents has been previously conducted in Spain. Indeed, we could not either find in the literature review any investigation regarding to the biological activity and quantification of major allergens in a set of different commercial extracts from B. tropicalis.

In our view, greater efforts should be made to implement a closer cooperation between allergen manufacturing companies and regulatory agencies to improve the quality and consistency of mite extracts not only in terms of comparing in-house reference standards but also among different companies. In the other hand, clinicians should bear in mind the limitations of currently available mite diagnostic extracts given that the types and levels of mite allergens that individuals are exposed to may differ to those contained in the commercially available extracts for SPT. Additionally, this study also serves as an inspiring attempt to develop “a la carte” instead of “one-size fits all” diagnostic extracts that can truly reflect the allergenic profile specificities of each population aiming for a better personalized medicine.