Standardization of house dust mite allergen extracts is essential for the development and improvement of diagnostic and therapeutic agents for house dust mite allergy patients. In Korea, almost all allergy diagnostics and immunotherapeutics are imported. This study may encourage researchers to initiate the manufacturing of diagnostic and therapeutic reagents in Korea.
A previous study produced house dust mite extracts using phosphate buffered saline. Its allergenic activity was evaluated using skin prick tests and intradermal skin tests.11
In this study, they achieved 81.0%-84.6% allergenic activity from Korean D. farinae
and D. pteronyssinus
extracts compared to commercially available skin test reagent extracts. Major allergen concentrations were measured to be 34.6 µg/mg for Der f 1, 17.0 µg/mg for Der f 2, 21.0 µg/mg for Der p 1, and 18.6 µg/mg for Der p 2.10
In the present study, bicarbonate buffer was utilized. Bicarbonate buffer led to enhanced extraction of allergenic molecules, especially Der f 1 (). Major allergen concentrations were also compared to US standardized extracts (). Although lower concentrations of major allergens were measured by two-site ELISA, the results were not found to be directly proportional to the in vitro
allergenic activity assayed by CAP inhibition tests or in vivo
assays. Sequence polymorphisms may partially explain the differences between the allergen content and skin test reactivities.12
It is necessary to investigate the sequence polymorphisms of major allergens. The difference in extraction procedures may also affect the concentrations of major allergens in the extracts. Notably, addition of phenol affected the protein concentration of allergen extracts ().
Recently, we studied sequence polymorphisms of Der f 1, Der p 1, Der f 2, and Der p 2, which may influence quantification, by using monoclonal antibody-based two-site ELISA kits.13
In a polymorphism study, Der p 2 variants with Asn114, which is responsible for the strong affinity to the monoclonal antibodies 1D8 (coating antibody in Indoor two-site ELISA kit) and 4G7 (detection antibody in indoor two-site ELISA kit) (currently 7A1 is being used), constitute 76.7% (46/60), and variants with Ser at position 47, which may have strong IgE affinity, accounted for 83.3% (50/60) from Korean D. pteronyssinus
isolate. The higher concentration of Der f 2 may be partly explained by decreased polymorphisms in antibody binding regions. For example, no sequence variation was found at position 114 of Der f 2 from the Korean house dust mite isolate. The lower levels of detected Der p 1 may have been influenced by the amino acid substitution at positions 124 and 182. This amino acid substitution may influence antibody binding, subsequently leading to decreased detection.
The allergenic activity of Korean and US allergen extracts was determined to be similar based on in vitro standardization. This observation is interesting because we observed some differences in major allergen content as determined by a commercial two-site ELISA kit (). These results imply that the actual concentration of group 1 and 2 allergens may be similar in these extracts. Western blot analysis of group 2 allergen using the monoclonal antibody 2F38 showed that there are similar concentrations of Der f 2 and Der p 2 in each mite extract, although there was stronger reactivity to Der f 2 than Der p 2 (). It is also possible that the allergenic components that do not belong to group 1 or 2 allergens could contribute to the overall allergenicity of the extracts.
Some components of the allergen extracts are known to influence immunologic and/or adjuvant activity.14
Chitin and β-glucan in house dust extracts may aggravate house dust mite allergen-induced allergic inflammation.15
Endotoxin is one of the most well characterized immunomodulatory substances contained in mite extracts. Due to the microflora residents in the mite gut, endotoxin is present in house dust mite extracts. Experimental studies have shown that exposure to low levels of endotoxin may exacerbate an allergic response; however, exposure to high levels might be protective.16-19
Epidemiologic studies have also shown that high endotoxin levels prevented the development of allergic disease in rural areas populated with stable animals. We compared endotoxin levels in Korean and US extracts (). Endotoxin contents measured in the extracts are within the range of those found in various US standardized dust mite extracts.20
extracts showed relatively high endotoxin concentrations, whereas D. pteronyssinus
extracts contained little endotoxin. This observation is in line with a previous report by Trivedi et al., in 2003.20
The difference in endotoxin concentration may reflect the different microflora in the mite gut. 16S rDNA fragments from multiple bacterial species were detected from house dust mite cultures.21
Bartonella species are thought to be the main source of endotoxins in house dust mite extracts.
These results suggest that researchers should choose the appropriate strain of house dust mite for their research aim. Further, removal of endotoxin from extracts may be necessary to accurately interpret results. Standardized allergen extracts produced in this study will be useful for the development of allergy diagnostics and immunotherapeutics in Korea.