Detection of anti-myelin antibodies in multiple sclerosis

NEIYrRALIZATION OF ANTI-MBP PURIFIED FROM CSF BY SELECTED SYNTHETIC PEPTIDES OF h-MBP K.G. W a r r e n and Ingrid Catz) MS Patient Care and Research Clinic, Dept. of Medicine, Univ. of Altmrta, Edmonton, CANADA. Htunan myelin basic protein (h-MBP) purified from central nervous system (CNS) myelin has a molecular weight of 18.5 KD and approximately 170 residues. Eighteen synthetic peptides ranging from 8 to 25 residues and covering the length of h-MBP were synthesized using the Fmoc method. Autoantibodies to h-MBP (anti-MBP) were isolated and purified from ccrebrospinal fluid (CSF) of patients with optic neuritis (ON) and multiple sclerosis (MS) by two-step atfinity chromatography. Purified anti-MBP was reacted with increasing amounts of each of the 18 synthetic peptides as well as hoMBP in an initial liquid phase, and then titers of free anti-MBP in the resulting mixtures were determined by a solid phase radioimmunoassay. Purified anti-MBP was neutralized by only 6 of the 18 synthetic peptides used in this study. The autoantibody was completely neutralized by peptides #12 (80-97), #15 (91-106) and #56 (75-95) and partially neutralized by peptides #16 (64-78), #21 (69-83) and #27 (61-75). The twelve remaining synthetic peptides covering both the carboxyi and amino terminals of h-MBP did not neutralize porified anti-MBP. These results suggest that anti-MBP purified from CSF of patients with MS and ON have affinity for discontinuous epitopes located between residues 61 and 106 on the h-MBP molecule. Alternatively anti-MBP may be polyspecifie recognizing different amino acid sequences.

DETECTION Of AI~FH-MYELIN ANTIBODIES IN MULTIPLE SCLEROSIS Jeffrey L Greenstein and Yentao Guo. Dept. of Neurology Temple University School of Medicine, Philadelphia, U.S.A. Multiple sclerosis (MS) is postulated to be an au~oimmune disease. Immune responses against a number of myelin proteins and glyeolipids have been detected but these are not specific to MS. To explore the potential repertoire of humeral responses to human myelin in MS, we developed a solid phase immunoassay capable of simultaenov~ly detecting an extensive range of myelin proteins. Normal human myelin was purified by density gradient centrifugation and solubilized using SDS. The proteins were separated on gradient SDSPAGE gels and transblotted onto nitrocellulose (NC). Proteins ranging from 14-300 K were adequately resolved. Known proteins wore identified by molecular weight determination and reaction with monoclonal antibodies. Western blot analysis of scra from MS patients and normal and other neurologic controls (OND: including stroke and head injuries) was performed using alkal;.ne phosphatase-conjugated polyelonal anti-human immunoglobulin and anti-human IgG, M and A antibodies. We det~:ed antibodies against both characterized and ,.mcbaracterized proteins ranging from 18-206 K in MS, normals and OND. Generally there was no specific pattern of reactivP,y for MS although occasional proteins only :eacted with MS scra; or reacted more strongly than OND and nnrmals. Antimyelin antibodies are, therefore, widely detectable in serum. Further analysis of CSF antibodies and cellular responses against identifiable proteins may be helpful in detecting relevant myelin antigens in MS.