Standardization of Weed Pollen Extracts, Japanese Hop and Mugwort, in Korea

Abstract

Purpose: Japanese hop (Humulus spp.) and mugwort (Artemisia spp.) are notable causes of autumn pollinosis in East Asia. However, Japanese hop and mugwort pollen extracts, which are widely used for the diagnosis, have not been standardized. This study was performed to standardize Japanese hop and mugwort pollen extracts.

Materials and methods: Allergen extracts were prepared in a standardized way using locally collected Humulus japonicus and purchased Artemisia vulgaris pollens. The immunoglobulin E (IgE) reactivities of prepared extracts were compared with commercial extracts via IgE immunoblotting and inhibition analyses. Intradermal skin tests were performed to determine the bioequivalent allergy unit (BAU).

Results: The IgE reactive components of the extracts via IgE immunoblotting were similar to those of commercial extracts. A 11-kDa allergen showed the strongest IgE reactivity in Japanese hop, as did a 28-kDa allergen in mugwort pollen extracts. Allergenic potencies of the investigatory Japanese hop and mugwort extracts were essentially indistinguishable from the commercial ones. Sums of erythema of 50 mm by the intradermal skin test (ΣED50) were calculated to be 14.4th and 13.6th three-fold dilutions for Japanese hop and mugwort extracts, respectively. Therefore, the allergenic activity of the prepared extracts was 90827.4 BAU/mg for Japanese hop and 34412 BAU/mg for mugwort.

Conclusion: We produced Japanese hop and mugwort pollen extracts using a standardized method. Standardized Japanese hop and mugwort pollen extracts will facilitate the production of improved diagnostic and immunotherapeutic reagents.

Keywords: Allergen standardization; Japanese hop; mugwort.

Fig. 1. Pollen allergen extraction procedure. Key steps and key points are summarized.

Fig. 2. In vivo standardization. Serially diluted allergen extracts were intradermally injected in subjects (A), and erythema size was measured after 15 to 20 minutes (B). The allergen potency was determined by calculating the dilution that induced an erythema diameter sum of 50 mm (C).

Fig. 3. SDS-PAGE (A) and IgE immunoblot (B) analyses of Japanese hop pollen extracts. Twenty microlligrams of proteins were separated on 15% polyacrylamide gel under reducing conditions. IgE reactive proteins were probed with a pooled serum of Japanese hop-sensitized patients. M, molecular weight standards; A, commercial (allergopharma) Japanese hop extract; Y, our (Yonsei) collected Japanese hop extract. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IgE, immunoglobulin E.

Fig. 4. SDS-PAGE (A) and IgE immunoblot (B) analyses of mugwort extracts. M, molecular mass marker; YS, Yonsei extract; HS, Hollister-Stier extract; A, mugwort-sensitized sera; B, non-atopic sera; C, buffer control.

Fig. 5. ELISA inhibition analysis of Japanese hop extracts. IgE reactivity to our (Yonsei) collected (A) and commercial (Allergopharma) (B) extracts was inhibited at a various concentrations. ELISA, enzyme-linked immunosorbent assay.

Fig. 6. In vitro standardization of mugwort extract. Competitive inhibition CAP was performed to estimate the potency of the extract using a pooled serum of eight subjects.