Streptavidin ap conjugate

The lectin reactivity of TF glycotope-specific antibodies was measured in a similar way, except that the binding of the neuraminic acid (sialic acid) specific Sambucus nigra agglutinin (SNA) to the absorbed anti-TF antibodies was determined as described earlier [37 (link)]. The biotinylated SNA (Vector Laboratories, Inc., USA) in 10 mmol/L Hepes, 0.15 mol/L NaCl, 0.1 mmol/L CaCl₂, pH 7.5 was applied at a concentration of 5 μg/mL for 1.5 h at 25°C. The bound lectin was detected with a streptavidin-AP conjugate (Dako, USA) and pNPP (Sigma, USA). The OD of control wells (no serum sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analysed in duplicate. The value of the SNA binding to all TF-specific Abs and the ratio of SNA binding to the level of TF-specific IgG, IgM, and IgA (SNA/Ig index) were determined.

The lectin reactivity of E2-specific IgG antibodies was measured in a similar way, except that the binding of the neuraminic acid (sialic acid)-specific Sambucus nigra agglutinin (SNA) to the absorbed anti-E2 antibodies was determined. The biotinylated SNA (Vector Laboratories, Inc., USA) in 10 mmol/L Hepes, 0.15 mol/L NaCl, 0.1 mmol/L CaCl₂, and pH 7.5 was applied at a concentration of 5 μg/mL for 1.5 h at 25°C. The bound lectin was detected with a streptavidin-AP conjugate (Dako, USA) and pNPP (Sigma, USA). The O.D. of control wells (no serum sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analyzed in duplicate. The value of the SNA binding to E2-specific Abs and the ratio of SNA binding to E2-specific IgG value (SNA/IgG ratio) were determined.

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).