Ab201795

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.

Paraffin-embedded renal sections of Thy-1N rats and MsPGN patients were deparaffinized in xylol and dehydrated in ethanol. Heat-induced tissue antigen retrieval in sections was performed using citrate buffer, and then endogenous peroxidase activity in tissues was blocked with 3% hydrogen peroxide. Next, non-specific antibody binding sites in tissues were blocked with 5% normal goat serum in PBS. The rat renal tissue sections were incubated with primary antibody against CCL3 (A7568), CCL4 (A1671), CD68 (ab31630), p-PKC-α (Thr638, ab32502), p-p65 (Ser536, sc-136548) and then incubated with HRP-conjugated anti-mouse IgG (BS12478). Some of these antibodies (A7568, A1671, ab32502) were conjugated with biotin by using a biotin conjugation kit (Abcam, ab201795) according to the instructions. The patient renal tissue sections were incubated with antibodies against p-PKC-α (ab32502), p-p65 (3033), IRF-8 (sc-365042), CCL3 (A7568), CCL4 (A1671) and CD68 (ab955), and then incubated with HRP-conjugated anti-rabbit IgG (BS13278) or anti-mouse IgG (BS12478). DAB staining was done, and the quantitative analysis of positive cell number or area was performed.

mAb-RA2 and mAb-RG2 antibodies were biotinylated using a biotinylation kit (Abcam; ab201795) according to the manufacturer’s instructions. In brief, 10 µL of biotin modifier reagent was added to 100 µL of each purified monoclonal antibody (2 mg mL⁻¹; PBS, pH = 7.4), followed by gentle mixing. The mixture was added to lyophilised biotin directly and mixed gently. Following 15 min at room temperature, the reaction was quenched by the addition of 10 µL of biotin quencher reagent.