Identification and characterization of a B-cell determinant within the amphipathic domain (residues 178-238) of the myelin proteolipid protein.
Pooled polyclonal rabbit anti-rat myelin and mouse anti-human proteolipid protein (PLP) antisera were screened against a panel of PLP synthetic peptides spanning residues 178-238 of the protein. Cross-reactivity against one determinant defined by PLP(200-219) was particularly prominent in both the anti-myelin and anti-PLP antisera and was chosen for further study. Competitive inhibition studies, utilizing a panel of overlapping synthetic peptides, demonstrated that the C-terminal portion of PLP(200-219), specifically residues comprising PLP(200-217), was important for antibody recognition of this region. Immunohistochemical analyses with an affinity-purified rabbit anti-PLP(200-219) antiserum demonstrated antibody cross-reactivity with PLP in both paraffin- and gelatin-embedded brain sections and immunocytochemical staining of mouse oligodendrocyte-enriched cultures demonstrated antibody binding with native PLP in situ. Staining of living non-permeabilized cells localized binding to the extracellular face of the myelin membrane. Collectively, these data argue for the presence of an immunodominant B-cell determinant defined by PLP residues 200-219. Furthermore, the structural conformation of this determinant in native PLP can be mimicked by the synthetic peptide, resulting in the generation of an antibody reagent that has considerable utility for immunohistochemical and immunocytochemical investigations of PLP expression and localization within the central nervous system myelin membrane.
Stephens
T S
TS
Memorial Research Center, University of Tennessee Medical Center, Knoxville, USA.
Pakaski
M
M
Lees
M B
MB
Potter
N T
NT
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007120:Immunochemistry; D007150:Immunohistochemistry; D051379:Mice; D008815:Mice, Inbred Strains; D018991:Myelin Proteolipid Protein / Q000276:immunology*; D011817:Rabbits
D000939:Epitopes; D007106:Immune Sera; D018991:Myelin Proteolipid Protein