1) ( Kalapothakis et al., 2002), L. laeta (SMase I, GenBank: AAM21154.1) ( Fernandes Pedrosa et al., 2002), and L. gaucho (A1H – LoxGa, GenBank: AAY42401.1) were prepared using the Spot technique ( Frank, 1992) according to the protocol described by Laune et al. (2002). In this study, however, a ResPep SL Automatic Spot synthesizer was used (Intavis AG, Bioanalytical
Instruments, Germany). Briefly, peptides were assembled using Fmoc chemistry on a cellulose membrane containing an aminopolyethyleneglycol moiety. The C-terminal residue of each peptide was coupled to the moiety. After Fmoc deprotection, the Epigenetics Compound Library high throughput other amino acids were sequentially added as in conventional solid-phase peptide synthesis. Finally the side chain protecting groups were removed by trifluoroacetic acid treatment in the presence of appropriate scavengers, while the linkage of the
peptides to the membrane was maintained. In the second series of experiments, three peptide sequences reactive to each species were selected and synthesized using the same conditions. For antibody binding studies, cellulose membranes were washed in 25 mM Tris-buffered saline containing 150 mM sodium chloride at pH 7,2 (TBS) and blocked overnight with 3% BSA in TBS and 0.1% Tween 20. The cellulose-bound peptides were subsequently washed and incubated at 37 °C with hyperimmune sera diluted in incubation buffer (1% BSA in TBS and 0.1% Tween 20) for 90 min. The diluted sera (1:5000 and 1:20 000) Tariquidar cell line were tested and the antibody binding was detected by adding peroxidase-conjugated anti-horse IgG antibody (Sigma; 1:30 000 dilution) for 60 min at room temperature. Following three 10-min washes in TBS (room temperature), spots were stained using a chemiluminescence detection ECL kit (GE Healthcare).
The membranes were used several times with sera of different neutralizing potencies previously determined on in vivo tests. Membranes were treated with a solution containing 8 M urea, 1% SDS and 0.1% β-mercaptoethanol for the removal of molecular complexes bound to the peptides. For the removal of urea, the membranes were washed with 50% ethanol and 10% acetic acid. To minimize peptide hydration, membranes were washed with methanol and subsequently dried and stored at −20 °C. These procedures allowed the re-use of the membranes without compromising their reactivity with antibodies. Based on the results obtained with ELISA-SPOT Roflumilast containing cellulose-bound peptides, the DNRRPIWNLAHMVNA-AGC peptide (Pep 1) and the DFSGPYLPSLPTLDA-AGC peptide (Pep 3) corresponding to residues 2–16 and 164–178, respectively, of the SMase I protein (L. laeta) were synthesized by Fmoc chemistry. Additionally, the EFVNLGANSIETDVS-AGC peptide (Pep 2), corresponding to residues 22–36 of the A1H-LoxGa (L. gaucho) and LiD1 (L. intermedia) proteins were also synthesized by Fmoc chemistry ( Laune et al., 2002). For the Fmoc chemistry, the ResPep SL Automatic Spot Synthesizer (Intavis AG, Bioanalytical Instruments, Germany) was used.