Control igg4

ELISA plate-bound Phl p 7 (0.1 μg/well) was pre-incubated with rabbit antisera (1:20 in buffer B) raised against Phl p 7, Phl p 7 peptides 1 or 2 (Fig. 4A) (inhibitors), or a normal rabbit serum (control). After washing, mAb102.1F10 or control IgG₄ (Sigma-Aldrich) (0.1 μg/well) was added and bound human IgG antibodies were detected with HRP-labelled rabbit anti-human IgG antibodies (Dako). All determinations were performed in duplicates, mean ODs were calculated and percent inhibitions were calculated according to the formula: 100 – (OD mAb102.1F10-binding after pre-incubation with inhibitors* 100/ OD mAb102.1F10–binding after pre-incubation with control) (Fig. 4B).

ELISA plates were coated with allergens (0.1 μg/well) and pre-incubated with mAb102.1F10 (2 μg/well) or with control IgG₄ (Sigma-Aldrich, 2 μg/well). Next, plates were incubated with sera from Phl p 7-allergic patients or a non-allergic subject (diluted 1:5). Bound IgE antibodies were detected with a mouse monoclonal anti-human IgE antiserum (PharMingen) followed by the HRP-labelled sheep anti-mouse antiserum (GE Healthcare). All determinations were performed in duplicates and mean values were calculated. Percent inhibitions were determined as follows: 100-(OD bound IgE after pre-incubation with mAb102.1F10 * 100/OD bound IgE after pre-incubation with control IgG₄) (Fig. 3).