As shown in Fig. GD1a, GT1a, and GD1c will also be elicited by LOS antigens of neuritis-causing strains (Aspinall 1994; Goodyear 1999; Koga 2005). Salloway (1996) reported GD3-like LOS (LOSGD3) inside a strain from a patient with Miller Fisher syndrome. In our earlier study, we shown that elevated titers of circulating antibodies to GD3 ganglioside [NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer] occurred in some individuals with inflammatory demyelinating polyneuropathies. We have identified in strain HS19 of the presence of an LOSGD3 having a tetrasaccharide epitope [NeuAc-NeuAc-Gal-Hep] (Usuki 2006) that has a terminal trisaccharide structure identical to GD3. This carbohydrate antigen causes LOSGD3-initiated nerve dysfunction in Lewis rats including interfering with ion channels essential for nerve conduction, and this is associated with improved anti-GD3 antibody (Usuki 2006). Recently, we have initiated development of gangliosidemimic therapy focusing on specific pathogenic antibodies with the goal of ameliorating the disease. This approach could prove superior to current GBS treatments, such as plasma-pheresis, intravenous administration of Ig, Lu AE58054 (Idalopirdine) and immunosuppressive chemotherapy; all of which target both pathogenic and non-pathogenic antibodies. In our first experiment, the efficacy of neutralizing anti-GD3 antibody by intraperitoneal administration of anti-idiotype monoclonal antibody BEC2 specifically directed to the anti-GD3 antibody (Usuki 2010) was examined. Our successful use of BEC2 to inhibit and neutralize circulating anti-GD3 and anti-LOSGD3 antibodies in the treated animals prompted us Lu AE58054 (Idalopirdine) to seek Lu AE58054 (Idalopirdine) additional and simpler epitope-neutralization therapies. Designing a 3D Lu AE58054 (Idalopirdine) conformational epitope structural mimic common to these carbohydrates and peptides, we decided to test using peptide mimics that can be synthesized easily for eventual clinical application. GD3-like peptides were selected by panning of a phage peptide library using an anti-GD3 monoclonal antibody (mAb) (Willers 1999; Popa 2006). In this study, we tested several phage-displayed GD3-like peptides for treatment of our established rat model of LOSGD3-induced neuropathy. The peptide treatment thus designed improved peripheral nerve function, and in this model was most likely a consequence of neutralizing and blocking the pathogenic activity of the elevated anti-LOSGD3/anti-GD3 antibodies. Materials and methods The following items were purchased: high-performance thin-layer chromatographic Lu AE58054 (Idalopirdine) plates coated with silica gel 60 (aluminum-backed linens) from E. Merck (Darmstadt, Germany), and a mouse hybridoma cell line for mAb R24 Jag1 from American Type Culture Collection (ATCC, Rockville, MD, USA). The GD3-like peptides were synthesized in the W. M. Keck Biotechnology Resource Center, Yale University (New Haven, CT, USA), based on the peptide sequences reported previously (Willers 1999; Popa 2006), shown in Table 1. The peptide sequences of PGD3-1, PGD3-2, PGD3-3, and PGD3-4 were initially reported by Popa (2006). Two other peptides, PGD3-5 and PGD3-6, were synthesized based on data reported by Willers (1999). Table 1 GD3-like peptide 1992). The nomenclature of gangliosides is based on that of Svennerholm (1964). Preparation of mAb R24 specific to GD3 See Appendix S1.1. Biotinylation of PGD3-4 One of the GD3-like peptides (PGD3-4) was biotinylated and used as bPGD3-4. See Appendix S1.2. Preparation of LOSGD3 ATCC-43446 (serotype HS19) was produced in broth with gentle shaking (100C150 rpm) for 48 h at 37C under microaerobic conditions. The cells were recovered by centrifugation at 7 000 for 30 min, and washed twice with saline. The LOS fraction (LS fraction) was extracted from the cell pellets by warm phenolCwater, followed by step-wise silica gel column chromatography (Usuki 2010). The LOSGD3 was purified from the LS fraction by mAb R24-linked affinity purification as previously reported (Usuki 2006, 2010). Pharmacokinetic analysis See Appendix S1.3. Streptavidin-coated ELISA for determination of plasma bPGD3-4 levels Plasma made up of bPGD3-4 was attached to streptavidin-coated 96-well polystyrene plates obtained from Pierce (No.15121, Rockford, IL, USA), and ELISA was performed according to the manufacturers instructions. The efficacy of binding of the anti-GD3 antibody to immobilized bPGD3-4 was decided using mAb R24, followed by an anti-mouse horseradish peroxidase-conjugated secondary antibody and colorimetric development. Briefly, the plasma samples from the single-dose administration of bPGD3-4 were applied to the streptavidin-coated ELISA plates at serial double dilutions in 1% bovine serum albumin/phosphate buffered-saline (BSA/PBS) answer. The plate was incubated for 1 h at room heat, and after washing with 1% BSA/PBS buffer, each well of the plate was treated with the mAb R24 (1 g/mL in 1% BSA/PBS buffer). After washing with 1% BSA/PBS.