Peptide reactivity between multiple sclerosis (MS) CSF IgG and recombinant antibodies generated from clonally expanded plasma cells in MS CSF  Xiaoli Yu, Don Gilden, Laura Schambers, Olga Barmina, Mark Burgoon, Jeffrey Bennett, Greg Owens  Journal of Neuroimmunology  Volume 233, Issue 1, Pages 192-203 (April 2011) DOI: 10.1016/j.jneuroim.2010.11.007 Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 1 Streamlined protocol to determine specificity of phage peptides. Individual phage plaque from 2nd and 3rd pans are amplified in U96 deepwell plates, and initially screened for positivity against panning antibody using ELISA (96 well single point ELISA). Potential positive clones were confirmed by duplicate ELISA/IPCR with isotype control rAb as negative control, followed by phage DNA purification and sequence analysis. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 2 ELISA/IPCR confirmed positive phage peptides. For ELISA (A and C), phage peptides were amplified (~1×109 pfu) and added to wells of ELISA plates pre-coated with panning rAb or IgG1 isotype control rAb from the same patient. Bound phage was detected by HRP-conjugated anti-M13 antibody followed by addition of HRP substrate ABTS for color development. Positive phage were those that had at least twice the OD value of the negative control. For IPCR (B and D), crude phage (~1×107pfu) from culture of U96 Deepwell plates were added directly to Protein A wells coated with panning rAb or BSA, and the bound phage was quantified by real-time PCR with M13 primers. Positive phages were those that had at least 1.4 CT less than that of the BSA control. The ELISA/IPCR were performed in duplicate and repeated at least once. Error bars represent standard deviation. Phage panned by two rAbs (#15 and #25) from patient MS02-19 are shown in Fig. A and C; phage by rAbs from patients MS03-7 and MS 04-2 are shown in Fig. B and D. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 2 ELISA/IPCR confirmed positive phage peptides. For ELISA (A and C), phage peptides were amplified (~1×109 pfu) and added to wells of ELISA plates pre-coated with panning rAb or IgG1 isotype control rAb from the same patient. Bound phage was detected by HRP-conjugated anti-M13 antibody followed by addition of HRP substrate ABTS for color development. Positive phage were those that had at least twice the OD value of the negative control. For IPCR (B and D), crude phage (~1×107pfu) from culture of U96 Deepwell plates were added directly to Protein A wells coated with panning rAb or BSA, and the bound phage was quantified by real-time PCR with M13 primers. Positive phages were those that had at least 1.4 CT less than that of the BSA control. The ELISA/IPCR were performed in duplicate and repeated at least once. Error bars represent standard deviation. Phage panned by two rAbs (#15 and #25) from patient MS02-19 are shown in Fig. A and C; phage by rAbs from patients MS03-7 and MS 04-2 are shown in Fig. B and D. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 3 Phage peptide binds to MS rAbs in a dose-responsive manner. MS rAbs (1μg/ml) from each of the 3 MS patient were coated onto wells of ELISA plates. Serial dilutions of corresponding phage peptides were added to each well. Bound phage was detected by HRP-conjugated anti M13 antibody followed by ABTS color detection. Irrelevant rAb from the same patient served as isotype control. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 4 Competitive inhibition ELISA confirms binding specificity of selected phage peptides. Phage peptides (~1×1010) were added to ELISA wells coated with anti M13 antibody. MS rAb (10μg/ml) pre-incubated with serial 3-fold dilutions of phage were added to each well. Bound rAb was detected by HRP-conjugated anti-Flag antibody. Binding of phage peptide to MS rAb in solid phase was inhibited by the same phage in solution, but not by an irrelevant phage peptide (4A: phage #25-D1 to rAb MS 02-19 #25; 4B: phage #10-B11 to rAb MS 03-7 #10; 4C: phage #13-C9 to rAb MS 04-2 #13). Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 5 Western blot demonstrates that some MS peptide epitopes are linear. All MS rAbs/CSF which selected positive peptides were used to evaluate bindings to linear peptides in Western blots. Shown are six phage peptides panned with both rAbs and CSF IgG from each of the three patients were separated on a gradient of 4–12% SDS polyacrylamide gel, blotted to a PVDF membrane and incubated with corresponding rAb and CSF IgG. Phage peptide D1 (lane 1), H3 (lane 2) and B11 (lane 3) bound specifically to panning antibodies rAb 02-19 #25, CSF02-19 IgG and rAb 03-7 #10, respectively (top panel, lanes 1, 2, 3), indicating that they are linear epitopes. No bands were seen with phage A2 (lane 4), C9 (lane 5) and E12 (lane 6) possibly because they may represent conformational epitopes. Anti-pIII antibody incubation with a duplicate membrane (lower panel) showed phage loading bands with all six phage peptides. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 6 Phage peptides selected by rAb and corresponding CSF IgG cross react. CSF IgG (5μg/ml) or representative MS rAb (1μg/ml) from patients MS 02-19 (A and B) and MS 03-7 (C and D), as well as inflammatory control CSF IgG were coated onto wells of protein A plates. Phage (109) panned by rAbs (A and C) were added to wells containing corresponding CSF IgG, and phage panned by CSF IgG (B and D) were added to wells containing representative rAb of the same patients. Bound phage was lysed by heating the ELISA plate at 95°C for 15min, and the phage number was determined by phage-mediated Immuno-PCR. All IPCR were performed in duplicate and repeated at least once. Fig. 6E shows that peptides panned by rAbs and CSF shared sequence homologies. Top panel: MS 02-19; Lower panel: MS 03-7. The identical residues are in bold and marked with symbol “*”. Symbols“:” and “.” indicate identical, conserved, and semi-conserved residues respectively. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 7 Phage peptides are specific for intrathecally-synthesized IgG in MS. MS CSF and paired serum IgG (5μg/ml) were coated onto wells of protein A plates. Phage (109) were added to the IgG coated wells, and bound phage was lysed and used as temples for real time PCR. A greater number of bound phage indicates a higher binding affinity. Peptides bound to MS CSF were 2.4 to 4 cycle thresholds (Ct) less than that to sera (one Ct equals to 2.2 fold difference), indicating that there were 5–10 times more phage bound to CSF than to sera. Pre-immune human IgG was used as a negative control. All immuno-PCRs were performed in duplicate and repeated at least once. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions

Fig. 8 MS rAbs bind to candidate protein peptides identified by database search. A. Four candidate peptides identified from a database search with phage peptide rAb 02-19 #25-D1 were synthesized by SPOT synthesis technology, and probed with rAb 02-19 #25 followed by anti-human IgG incubation and chemiluminascent detection. Peptides derived from candidate proteins plexin domain-containing protein 1 and glycogen synthase were recognized by rAb 02-19 #25. Sequence alignments are shown on the right. A star (*) indicates identical amino acid residues and a semicolon (:) indicates conserved amino acid residues. B. lists candidate peptide sequences testing for binding to rAb #25. Journal of Neuroimmunology 2011 233, 192-203DOI: (10.1016/j.jneuroim.2010.11.007) Copyright © 2010 Elsevier B.V. Terms and Conditions