PURPOSE: In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome. METHODS: Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined. RESULTS: Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope. CONCLUSIONS: The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.
Allergens ; Animals ; Apiaceae ; Apium graveolens ; Artemisia ; Bacteriophages ; Betula ; Carbohydrates ; Clone Cells ; Enzyme-Linked Immunosorbent Assay ; Food Hypersensitivity ; Glycoproteins ; Horseradish Peroxidase ; Humans ; Immune Sera ; Immunization ; Immunoglobulin E ; Immunoglobulin G ; Mice ; Molecular Weight ; Pollen ; Spices ; Vaccination ; Virtues

OBJECTIVETo develop a reliable method to assess the stability of xinyue capsules containing Panax quinquefolius saponins according to European quality standards.

METHODSAn efficient high-performance liquid chromatography ultraviolet (HPLC-UV) method was established to analyse six main ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in six different batches (120 capsules/batch) from the same lot of xinyue capsules and in one batch measured six times within one day. The six ginsenosides were separated on a Hypersil BDS-C18 column (3 μm, 100 mm×3 mm) at a flow rate of 0.5 mL/min. Gradient elution was performed using a mobile phase gradient of acetonitrile-water modified with 0.01% formic acid. The HPLC chromatograms were analyzed with "LC data comparison" using Lab Solutions software.

RESULTSThe HPLC peaks were identified by comparing their retention times (Rg1: 23.44 min, Re: 23.77 min, Rb1: 35.24 min, Rc: 36.18 min, Rb2: 38.55 min and Rd: 40.88 min) with those of the standards under the same chromatographic conditions, which showed similar results among the samples of six different batches and among the samples from one batch detected six times within one day.

CONCLUSIONSXinyue capsules have good drug intra-day consistency at room temperature and exhibit a consistent quality between different batches. This study established a reliable method to assess the stability of xinyue capsules, which is suitable for further qualitative analysis and may assist in promoting the safe and effective use of Chinese herbal medicine.

Capsules ; Chromatography, High Pressure Liquid ; methods ; Drug Stability ; Ginsenosides ; analysis ; Saponins ; analysis