Current Status of Standardization of Inhalant Allergen Extracts in Korea

Allergy diagnosis and immunotherapy in Korea rely mostly on imported allergen extracts. However, some allergens that are not important in Western countries are not commercially available, and even the same species of allergen source often displays differences in allergenicity due to amino acid sequence polymorphisms. Therefore, it is essential to prepare allergen extracts that reflect regional characteristics. Allergen standardization has been performed since 2009 with the support of the Korea Center for Disease Control and Prevention. Here, we summarize the current status of allergen standardization, focusing on the house dust mite and cockroach. Pollen allergens that are under investigation are also briefly described.


INTRODUCTION
http://e-aair.org Evaluation and Research (CBER, Food and Drug Administration, USA) ( Fig. 1). Interestingly, compared to the CBER reference, the Korean reference material showed only 11.6% allergenic potency in terms of 50% inhibitory concentration, and the commercial extract (HollisterStier, Spokane, WA, USA) showed 14.6% potency. In addition to allergen potency, allergen composition is also important in terms of the consistency of allergen preparations. However, the concentration of major allergens of the house dust mite is known to be variable, even in commercial skin test reagents. 7,8 For example, the ratio of Der p 1 (12-30 μg/mL)/Der p 2 (3-15 μg/mL) ranges from 1.1:1 to 6:1. 7 Mite extracts prepared from raw materials rich in fecal pellets contain higher ratios of mite group 1/2 allergens than those from pure mite bodies. 9 In the extracts from Korean house dust mite isolates, we measured concentrations of 5.0 μg/mg Der f 1, 12.0 μg/mg Der f 2, 11.6 μg/mg Der p 1, and 12.4 μg/mg Der p 2 by 2-site ELISA (Indoor Biotechnologies Inc., Charlottesville, VA, USA). 6 Quantification of mite allergens, particularly group 2 allergens, is known to be influenced by amino acid sequence polymorphism. 10 Sequence polymorphism analysis of Korean mite isolates revealed the predominant and novel variants of mite group 1 and 2 allergens. 11  No. JN222805-9. Moreover, most of the variants contained Ser47 which is known to have a higher IgE-binding affinity, and Asn114 which is responsible for strong binding to the monoclonal antibodies 1D8 and 4G7 in the commercial ELISA kit. In a study from Thailand, rabbit polyclonal antibodies were used for the quantification of Der f 1. 12 The problems caused by polymorphisms may be overcome by the production of a regional standard using a mixture of predominant variants and/or development of monoclonal antibodies recognizing non-polymorphic regions. Stability of mite extracts have also been investigated. 13 Storage temperature is the most important factor in the preservation of allergenicity of the extract, and 92.0%-97.0% of IgE reactivity is retained at 1 year regardless of buffer composition when stored at 4°C. The mite extract reconstituted in 50% glycerol retained more than 89.9% IgE reactivity at 1 year even at room temperature.

Cockroach
Cockroach is another important source of indoor allergens associated with asthma exacerbation. 14 However, cockroach extract has not been standardized, and manufacturers commonly use the unit of weight (raw material) to volume (extraction buffer) (v/w). Strong protease activity in the cockroach extract makes it difficult to standardize the extract. 15 We prepared an extract of German cockroach and analyzed its allergenic properties. 16 The cockroach population used in this study originated from a single pregnant female captured in Seoul. 17 The cockroach extract showed allergenic potency (94.2%) similar to the commercial extract (Hollister-Stier) in a competitive IgE-binding analysis. However, there was a substantial difference in Bla g 1 and Bla g 2 concentrations between the extracts: the Korean extract contained 404 U/mg Bla g 1 and 273 ng/mg Bla g 2, whereas the Hollister-Stier extract contained 187 U/mg Bla g 1 and 56 ng/mg Bla g 2. A difference in endotoxin concentration between the extracts was also observed (3,440 EU/mL for Korean extract and 6,580 EU/mL for HollisterStier extract); however, endotoxin is not considered essential 18 even though alveolar macrophages play a key role in a cockroach-induced airway inflammation 19 in a mouse model. Interestingly, proteins of approximately 60 and 70 kDa showed strong IgE reactivity in both extracts. Proteomic analysis of the extract showed that 60 and 70 kDa allergens are α-amylase and Bla g 3, respectively (Fig. 2, Table 1). Amylase was also identified as an important allergen in German cockroach fecal extract. 20 An interesting finding of fecal extract analysis is the IgE reactivity of various digestive enzymes, such as α-amylase, trypsin, chymotrypsin, metalloprotease, and midgut carboxypeptidase A. Furthermore, we detected glycinin-like protein, which may have been derived from the cockroach diet and may lead to false diagnosis. Further studies on α-amylase and Bla g 3 allergens are needed because they appear to have various isoforms.

Standardization of pollen allergens
Pollens from various plants are important causes of seasonal allergic rhinitis. Tree pollens from alder, birch, and oak are known to be important in spring, mainly from March to May, whereas weed pollens from Japanese hop, sagebrush, and ragweed are important in autumn, mainly from August to September. 21 Currently, most of the commercial tree and weed pollen extracts are not standardized and are provided as a protein nitrogen unit (PNU) or weight per volume unit (w/v, 1:10 to 1:40). The recommended maintenance dosage for immunotherapy, the probable effective dose range, was estimated to be 0.5 mL of 1:100 or 1:200 w/v dilution of the non-standardized pollen extract. 5 For short ragweed, a maintenance dose of 6-12 μg Amb a 1 or 1,000-4,000 AU was calculated for effective treatment. Furthermore, most of the pollen extracts are imported from Western countries. Therefore, pollen extracts from trees that are not common or not even found in Korea are currently being used for allergy diagnosis and immunotherapy. A correct diagnosis of pollen allergy is essential because sensitization to pollen allergens may lead to allergic reactions to various fruits. 22

Tree pollen
The levels of airborne pollens from pine, oak, and birch were reported to be high in May in Korea. 23 Moreover, the sensitization rates to tree (alder, pine, and oak) and weed pollens (Japanese hop and mugwort) have increased significantly over 10 years (1999 to 2008) in the southern part of Gyeonggi province. 24 In particular, common silver birch (white birch, Betula verrucosa) and white oak (Quercus alba) are not native to Korea. How-ever, Siberian silver birch (B. platyphylla var. japonica) is often planted as a landscape tree. 25 In the case of oak species, Mongolian oak (Q. mongolica) is known to be a predominant species in Korea, 26 whereas sawtooth oak (Q. acutissima) is often found only at human dwellings. Pathogenesis-related 10 proteins (PR-10, Bet v 1 homologues) from the order Fagales are known to be important allergens, but cross-reactivity could be limited. 27 Therefore, there is an urgent need to determine crossreactivity between pollen extracts from trees native to Korea and the commercially-available extracts from foreign trees. A preliminary study suggested that sawtooth oak is a major cause of oak allergy in Korea. Therefore, we are characterizing the PR-10 protein, a putative Que ac 1, from sawtooth oak and the potency of the sawtooth oak pollen extract is being evaluated by an in vivo method.

Weed pollen
Pollens from Japanese hop, sagebrush, and ragweed constitute about 90% of weed pollens in the air in Korea during autumn. 28 In particular, 2 invasive plants, Japanese hop (Humulus japonicus) and ragweed (Ambrosia artemisiifolia var. elatior), have become able to displace native species as a result of global warming. 29 Most importantly, Japanese hop pollen extract is not standardized despite its allergenic importance in East Asia. A study from China reported that profilin (a putative Hum j 2) is a major allergen from the Japanese hop. 30 However, 2 isoforms of recombinant profilin were found to play a minor role in Korea. 31 An allergen with a molecular mass of approximately 11 kDa is believed to be a major allergen. 32,33 We are currently attempting to characterize this allergen of interest. The potency BAU of the allergen extract is also being determined by an intradermal skin test. Detailed information on in vivo standardization and characterization of 11 kDa allergen of Japanese hop will be published elsewhere.
At CBER, the potency of the ragweed allergen extract is evaluated through determination of a specific allergen, Amb a 1 by immunodiffusion with sheep polyclonal antibodies. However, a commercial 2-site ELISA kit (Indoor Technologies Inc.) uses monoclonal antibodies for the quantification of Amb a 1. Therefore, it is necessary to compare these 2 methods.
Artemisia vulgaris is commonly used for the diagnosis of mugwort allergy in Korea. However, this species is not found in Korea, and A. princeps is reported to be a dominant species. 34 Comparison of 6 different species of Artemisia (A. vulgaris, A. sco-  The data are summarized in Table. A B  35 Therefore, we are conducting in vivo standardization of mugwort extract using A. vulgaris.

Recombinant allergens
The concentration of major allergens could be affected by environmental stresses. The mite culturing diet 36 and the temperature at which the mites are cultured 37 are known factors that determine allergen concentrations in extracts. The expression of cockroach allergen Bla g 1 is known to be influenced by food intake, 38 whereas excretion of Bla g 2 is increased after exposure to a sublethal dose of boric acid, a common pesticide. 39 Glutathione S-transferases, mite group 8 and cockroach group 5 allergens, are thought to be influenced by environmental chemicals because of their detoxification activity. 40,41 A similar situation exists for pollen allergens. Pollen production can be increased by exposure to environmental pollutants, high CO2 concentrations, and high temperatures. 42 High-quality recombinant allergen should ensure the absence of non-allergenic antigens and adjuvant-like molecules, such as endotoxin, β-glucan, superantigen, and chitin. Even if high-quality allergen extracts are available, production of recombinant allergens is desirable for the quantification of major allergens. Quantification of major allergens is often hampered by polymorphisms. Therefore, it is necessary to identify dominant isoforms and produce regional standard molecules. Currently, various recombinant allergens from house dust mite, cockroach, Asian needle ant, buckwheat, and pollens are being characterized for standardization.

Concluding remarks
Allergen standardization is performed not only by the measurement of IgE-binding potency but also by the quantification of major allergens. 43 However, many of the major allergens from Korean trees and weeds have not been characterized. Furthermore, the clinical value of component-resolved diagnosis has been proven. 44 Continued effort is needed to produce validated recombinant allergens and to develop a quantification system for better standardization. 45 Finally, allergen standardization could facilitate the development of advanced diagnostic and immunotherapeutic reagents.