Materials and methods Characterization of the cattle allergic farmers The sera of 42 farmers (26 male, 16 female; age 25–74, mean 52.2, median 52 years) with cattle-related Selleck GDC-0994 symptoms (29 upper airway symptoms such as allergic rhinitis, 37 asthmatic symptoms, 19 skin symptoms such as itching, eczema and urtica) were investigated. Most of the farmers kept cattle races such as Holstein-Friesian (HF, n = 23), mainly in the northern parts of Germany; in the southern parts of Germany, the main cattle races were German Simmental (GS, n = 15) and German Brown (GB, n = 14). Only a few farmers kept races uncommon to Germany such

as German Red Pied (GRP, n = 7), Charolais (Ch, n = 5), Blonde Aquitaine (BA, n = 2), Jersey (J, n = 1), or Limousin (L, n = 1). Additionally, two non-farming control subjects who had never shown allergic symptoms or reactions against animal-derived antigens were included in the study. The detection of specific learn more IgE antibodies was performed using CAP RAST® (CAP-System, Pharmacia Diagnostics, present name: Phadia, Freiburg, Germany). Commercial cow allergen extracts Raw material from four different manufacturers of skin test extracts (Allergopharma, Reinbek near Hamburg, Germany; ALK-Scherax, Hamburg, Germany; Bencard, Munich, Germany; HAL,

Düsseldorf, Germany, hereafter referred to as A, B, C, and D, respectively) was used. After reconstitution of the lyophilized raw material in distilled water, the total protein content was about 4 mg/ml. Self-made cow allergen extracts Cattle selected for this study were all healthy to avoid a possible influence of pathologic conditions on the cattle allergen production. Farmers were instructed to cut the cattle hair close to the skin without visible contamination. The hair of cattle of different breeds was used, including www.selleck.co.jp/products/Gemcitabine(Gemzar).html samples of the most common cattle breeds in Germany, namely Holstein-Friesian, German Brown, Limousin, Charolais, German Simmental, Blonde d’Aquitaine and German Red Pied. Two grams

of hair were extracted with 20 ml of 0.125 M NH4HCO3 for 24–72 h at 4°C, following centrifugation. An incubation period of 44 h was found to yield optimum results in protein content and SDS-PAGE separation (data not shown). Protein determination Protein content was determined using the bicinchonic acid procedure as described by Pierce Chemicals, Rockford, USA. The results were verified using different dilutions of each sample. The samples were lyophilised and reconstituted in 10% of the original volume, then stored at −20°C. It was verified that the lyophilized extracts did not show any differences concerning total protein content or SDS-PAGE analysis compared to the unlyophilized extracts (data not shown). SDS-PAGE/immunoblot The detection of the allergenic proteins in the extracts was performed by immunoblotting.