Immunoblotting was performed to (1 (link)) demonstrate IgE sensitization of Iranian patients recruited by clinical history (not shown) and (2 (link)) to assess the IgE sensitization pattern for selected patients lacking detectable IgE binding to purified Periplaneta allergens. Briefly, P. americana extract (50 μg/cm) was applied to SDS–PAGE, transferred to nitrocellulose membrane (0.2 μm, Amersham Protean, GE Healthcare, Freiburg, Germany) by semi-dry blotting, and visualized by Ponceau S staining (Sigma-Aldrich, Munich, Germany). After blocking with 0.3% Tween-20 in Tris-buffered saline (TBS, 50 mM Tris, 150 mM NaCl, pH 7.4), the membrane was incubated overnight with patient's serum (500 μl/strip, diluted 1:10 in TBS, 0.05% Tween-20, 0.1% BSA), and bound IgE was detected as described elsewhere (27 (link)).
Specific IgE values to Periplaneta (i206) and Blattella extract (i6) were determined by ImmunoCAP tests (Thermo Fisher Scientific) and to single recombinant Periplaneta allergens by experimental ImmunoCAP tests as previously described (28 (link)). Assay background of each test was assessed and cutoff level for positivity adapted as required (0.35 kUA/L for Per a 2, Per a 5, and Per a 10 and 0.10 kUA/L for all other allergens).
Specific IgE values to Periplaneta (i206) and Blattella extract (i6) were determined by ImmunoCAP tests (Thermo Fisher Scientific) and to single recombinant Periplaneta allergens by experimental ImmunoCAP tests as previously described (28 (link)). Assay background of each test was assessed and cutoff level for positivity adapted as required (0.35 kUA/L for Per a 2, Per a 5, and Per a 10 and 0.10 kUA/L for all other allergens).