Mouse anti human ige biotin conjugate

Allergenicities (IgE binding frequencies) of the nGST and the rGST were determined by IgE-ELISA. The assay was performed as described previously^{17 (link)}. The nGST and rGST (5 μg/ml carbonate-bicarbonate buffer, pH 9.6) were added to separate wells (100 μl/well) of a microtiter plate (Costar, MA, USA) and kept at 37 °C until dried. All GST-coated wells were washed and blocked with 200 μl of a blocking solution (1% BSA in PBS) before adding with serial two-fold dilutions of individual sera and the plate was incubated for 3 hours. Wells added with only the serum diluent served as blank. All wells were washed and added with 100 μl of mouse anti-human IgE-biotin conjugate (Southern Biotech, AL, USA; diluted 1:1,000 in PBS-T). Streptavidin-horseradish peroxidase (HRP) conjugate (Dako Cytomation) and ABTS substrate solution (KPL) were used for color development. Absorbance at 405 nm (OD_405nm) of the content in each well was determined (ELISA reader, MultiscanEX, Labsystem, Helsinki, Finland) against the blank. Cut-off OD_405nm between positive and negative IgE-ELISA was arbitrarily set at ≥mean OD_405nm of non-allergic sera + 2 standard deviations (SD).

Isoforms of nGST were determined by a gel-based proteomics. Purified nGST was subjected to 2DE as described previously^{39 (link)}. For the first dimensional electrophoresis, 7 cm-IPG strips and 0.5% pH 3–10 IPG buffer (GE Healthcare) were used. The electrophoresed-IPG strips were then subjected to 12% SDS-PAGE and proteins in the gel were stained by CBB. Gel pieces containing proteins of ~21 kDa were excised from the stained gel and subjected to in-gel tryptic digestion and LC-MS/MS, respectively. Protein orthologues were identified by comparing the peptide sequences of the P. americana-GST generated from the mass spectrometry with the Arthropoda/Insecta database sequences.
The nGST isoforms were checked for their reactivity to IgE in the pool of CR allergic patients’ sera by 2DE IgE-immunoblotting. The 2DE-separated nGST was electro-transblotted onto an NC, blocked with BSA, and the blot was allowed to react with the CR allergic patients’ serum pool. After keeping at 4 °C overnight, the NC was washed with TBS-T before placing in a solution of appropriately diluted mouse anti-human IgE-biotin conjugate (Southern Biotech) and kept at 25 °C on a rotating platform for 3 hours. Spots of the nGST isoforms bound by the specific serum IgE were revealed by using streptavidin-AP conjugate (Dako Cytomation) and BCIP/NBT substrate (KPL).