Montage antibody purification kit

Rabbits were immunized subcutaneously with three 500 μL doses of recombinant P33 (50 μg/dose) in two-weeks intervals. Before immunization and two weeks after the third immunization, blood was collected, and IgGs were purified from the serum using a Montage Antibody Purification Kit and Spin Columns with PROSEP-A Media (Millipore, Billerica, MA, USA). The paraffin was removed from salmon louse sections with xylene and then hydrated by successive 2 min washes with a graded series of 100%, 95%, 80%, 75%, and 50% ethanol. The slides were treated with Proteinase K (Dako, Barcelona, Spain) for 7 min, washed with PBS, and incubated with 3% BSA (Sigma–Aldrich) in PBS for 1 h at RT. Then the slides were incubated with anti-P33 rabbit serum diluted 1:100 in 3% BSA/PBS for 14 h at 4 °C. After additional washes in PBS, the sections were incubated with FITC conjugated goat anti-rabbit IgG secondary antibodies (Sigma–Aldrich, St. Louis, MO, USA), diluted 1:80 in 3% BSA/PBS, for 1 h at RT. Finally, the slides were mounted using Prolong Gold antifade reagent with DAPI reagent (Molecular Probes, Eugene, OR, USA). The slides were examined using a Zeiss LSM 800 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). Sections incubated with pre-immune serum were used as controls.

POM1, POM3, POM4, POM6 and POM11 antibodies (Polymenidou et al., 2008 (link)) were provided to J.T. by the University of Zürich, Institute of Neuropathology. Hybridomas producing the human-mouse chimeric monoclonal antibodies D13 and D18 (Safar et al., 2002 (link); Williamson et al., 1998 (link)) were provided by Anthony Williamson, Dennis Burton, and Bruce Chesebro. Antibodies were affinity-purified using protein A spin columns (Montage Antibody Purification Kit, EMD Millipore). Fab fragments of D18 were prepared using the Pierce Fab preparation kit from Thermo Scientific. The purity of all antibody preparations was verified by SDS-PAGE. The following antibodies were purchased from commercial sources: 100B3 (Wagening UR, Netherlands); 6D11 (BioLegend, cat #808001); ICSM-18 and ICSM-35 (D-Gen Ltd.).
Pentosan polysulfate (average MW = 4500–5000) was purchased from Biopharm Australia Pty Ltd., and phosphatidylinositol-specific phospholipase C from Sigma (cat #P5542).

Hybridoma cells producing a monoclonal antibody (designated C166) was derived to the nitrated peptide AC-KEKKCS(YNO₂)TED-NH2 sequence from Apolipoprotein H a protein in the same complement control superfamily as CFH using their proprietary technology (Abmart, Berkeley Heights, NJ). For production of purified monoclonal antibodies, hybridoma cells were cultured in culture media DMEM/F12 containing 5% FBS. When cell density reached 1×10⁶/ml, the supernatant was collected after 12 h incubation in serum free medium. The C166 monoclonal antibody in the supernatant was purified using the Montage Antibody Purification Kit (Merck Millipore). The concentration of the purified C166 anti-nCFH monoclonal antibody was measured by Nano-drop 2000c (Thermo Fisher).