Af1674

Our ELISA detecting Abs to OMGP used recombinantly produced OMGP, which was enzymatically biotinylated on its Avi-tag. Further details and ELISAs to detect MOG-Ab h8-18C5 and to quantify C1q-binding of OMGP-specific Abs are in the Additional file 1. We developed an ELISA to measure soluble OMGP. To this end, we used our new anti-OMGP mAb 14A9 for coating, rat rIgG2b (BD Biosciences, 556968) was the control Ab. For detection, we used the polyclonal OMGP antibody (R&D, AF1674), which we had biotinylated with a biotinylation kit (abcam, ab201795). To detect OMGP in CSF, samples were diluted 1:30 in PBST-0.5% BSA.

CSF samples were pooled and approximately 30 × concentrated using filter columns (Amicon 3 kDa). These, together with recombinant OMGP and immunoglobulins were separated by SDS gel (Invitrogen, NP0321) and transferred on an activated (10% methanol) PVDF membrane (GE Healthcare, 10600023). As primary Ab, goat anti-OMGP (R&D, AF1674) was used. As secondary Ab, donkey anti-goat-IgG-HRP (Invitrogen, A16005) was used, because it shows only minimal cross-reactivity with human IgG due to absorption against human IgG. ECL prime solution (GE Healthcare, RPN2232) was used and signal was detected by digital imaging systems Odyssey Fc from Leica.