PMC

Array analyses of CSF and serum. Heat maps created using logarithmic transformations of the mean intensities recorded for each lipid antigen depict CSF (A) and serum (B) IgG reactivity in MS and OND patients. Lipids are displayed as column headings and each row represents an individual patient. (C) Anti-sulfatide/sulfatide complexes in CSF of patients with MS and OND. Closed circles denote MS CSF and open circles denote OND CSF. Reactivities to sulfatide and particular combinations of sulfatide and other lipids were observed more frequently within MS CSF (p values were obtained by ANOVA with Bonferroni correction).

Illustrative blots and their quantitative analysis. Data were obtained using MS CSF (A–D) and recombinant antibodies 3, 73, 17 () cloned from a single CSF-resident plasma cell (E–J). Row and column headings reveal the complex at each location. We used the following lipids either alone or in combination: sulfatide (Sul), galactocerebroside (GalC), ceramide (Cer), cardiolipin (Card), sphingosine (SS), sphingomyelin (SM), cholesterol (Chol), digalactosyl diglyceride (DGG), monogalactosyl diglyceride (MGG), phosphatidylcholine (PC). “Xs” represent the negative controls (methanol) which act as a line of symmetry for duplicate spots on the same membrane. (A, C) PPMS CSF blot and quantitative data, for binding to the complex formed by cholesterol and cardiolipin is increased by 96.3% compared to the sum of the intensities for the individual lipids (p = 0.0004, paired t test n=3). (B, D) Complex attenuation in a CSF MS blot, where binding to sulfatide yields a mean relative intensity signal of 57.1%, yet the intensity for the complexes of sulfatide created with the lipids ceramide, sphingomyelin and phosphatidylcholine is almost eliminated at between 1.96% and 3.66% (p<0.0001, GLM ANOVA with Tukey n = 3). (E, G) Blot and quantification from MS rAb 3 with anti-myelin lipid complex reactivity. Binding to the complex of sulfatide and galactocerebroside is increased by 82.84% (p = 0.004 paired t test, n = 3) compared to the sum of the mean intensities recorded from the individual lipids. (F, H) Blot and quantification from MS rAb 73 with binding to sulfatide that is inhibited when sulfatide is complexed with the lipid sphingomyelin (p<0.0001 GLM ANOVA with Tukey, n = 3). (I, J) MS rAb 17 assayed by serial dilution. The pattern of binding seen at higher rAb concentrations when signals are saturated appears largely complex independent. At a lower concentration of 0.1 µg/ml binding to the complex created by sulfatide and galactocerebroside can be seen to be enhanced (p = 0.0143 paired t test, n=3).

Array analyses for rAbs. (A) Heat map depicting reactivity of rAbs derived from MS and OND patient CSF. The lipid antigens are displayed as column headings and each row represents an individual rAb. lists the sources of MS and control rAbs and more detailed analyses of individual rAbs can be found in and . Covariance clustering of all rAb reactivities has been performed revealing two populations of lipid reactive antibodies; one directed against sulfatide or sulfatide/lipid complexes and the other against cholesterol. MS rAbs do not segregate with unique binding profiles, as lipid reactivities are also seen in OND rAbs (19% V 37%). (B) Blot of the MS derived rAb 76. Note antibody binding to sulfatide is virtually abolished in the presence of sphingomyelin. (C) Blot of the mAb O4. In contrast to rAb 76, binding to sulfatide is enhanced not inhibited by sphingomyelin. (D) Unfixed myelinating cultures derived from embryonic rat spinal cord were stained at 4 °C with either (D) (i)–(iii) MS derived rAb 76 (10 µg/ml) or (D) (iv)–(vi) mAb O4 (10 µg/ml) to visualize sulfatide (green) and the MOG-specific mAb Z2 (10 µg/ml) to identify oligodendrocytes and myelin sheaths (red). After fixation with 4% paraformaldehyde bound antibody were detected using species-specific secondary reagents (anti-human — green; anti-mouse — red). Control cultures were stained with polyclonal human IgG, mouse myeloma proteins and/or secondary antibodies alone. In no case did immunoreactivity for MOG (red) at the surface of oligodendrocytes (arrow head) or myelinated internodes (small arrows) co-localize with bound human rAb ((ii), green) as demonstrated in the merged image (iii). Staining obtained using this and other rAbs was restricted to a diffuse background indistinguishable from that observed when cultures were stained using polyclonal human IgG pooled from multiple donors. In contrast the sulfatide reactive mAb O4 binds the surface of oligodendrocytes and myelin sheaths where it co-localizes with the MOG-specific mAb Z2 (iv)–(vi). This marked difference in immunoreactivity of sulfatide reactive rAbs and mAb O4 is associated with marked differences in recognition of lipid complexes using lipid arrays.