Protein g column

The four bivalent monoclonal IgG
antibodies and proteins used in the experiments were designed, expressed,
and purified according to earlier published work.^{6 (link),21 (link)} The
RmAb158 monoclonal antibodies selectively bind to Aβ protofibrils,^{22 (link)} whereas the RmAb2G7 monoclonal antibodies selectively
bind to high-mobility group box 1 (HGMB1) proteins.^{23 (link)} In short, the heavy and light chain scFv8D3 transferrin
receptor transporter variable region sequence^{20 (link)} was connected to the C-terminus of the RmAb2G7 or RmAb158 light
chain with in-house designed linkers (APGSYTGSAPG or APGSGTGSAPG,
respectively). Figure 2 shows the cartoon representations of the antibody design, showing
the location of conjugated scFv8D3 in the modified antibodies.
The four recombinant antibodies were expressed
using Expi293 cells
(Thermo Fisher) transiently transfected with pcDNA3.4 vectors using
polyethyleneimine (PEI) as the transfection reagent. All antibodies
were purified on a protein G column (Cytiva) and eluted with an increasing
gradient of 0.7% acetic acid. The buffer was exchanged for phosphate-buffered
saline (PBS) (Gibco) immediately after elution, and the protein concentration
was determined at A280.

Fabs were expressed in TBG medium for 22 hours at 30 °C. Cell pellets were lysed using a high-pressure homogenizer in running buffer (20 mM sodium phosphate pH 7.0) containing 1 mg/mL hen egg lysozyme (Sigma). The supernatant, after centrifugation, was loaded onto a protein G column (Cytiva) pre-equilibrated with running buffer. Fab was eluted using 100 mM glycine (pH 2.7) and immediately neutralized using 2 M Tris buffer (pH 8.0). The eluted fractions with Fab were pooled, flash-frozen, and stored at −80 °C until further use.

For ordinary antibodies, the sequences of their variable region were codon-optimized and synthesized in Azenta and then inserted into pcDNA3.4 vector plasmid containing human IgG1 heavy chain constant region sequence or light chain constant region sequence (κ or λ chain). To express these antibodies, 15 μg heavy chain plasmid and 15 μg light chain plasmid of the same antibody were cotransfected into 30 mL Expi293F cells using ExpiFectamine™ 293 Transfection Kit (Thermo Fisher) according to the manufacturer’s instructions. The Expi293F cells were then cultured for 120 h in a shaker under 37 °C and 5% CO₂. The expressed products were centrifuged at 4000× g at 4 °C for 20 min, and the supernatants were filtered through a 0.22 μm filter. The filtered supernatant was then subjected to ProteinG column (Cytiva), equilibrated with phosphate-buffered saline (PBS), pH 7.4. After washing with 5 column-volume (CV) PBS, the antibody was eluted with 5 CV 0.1 M Glycine, pH 2.7 and immediately neutralized to pH 7.4 using 1 M Tris-HCl, pH 9.0. The eluted antibodies were then exchanged into PBS and concentrated using a 30 kDa ultrafiltration centrifugal tube. The concentration of purified antibodies was measured using Nanodrop Plus (Thermo Fisher). The antibodies were then aliquoted and stored at –80 °C until use.

Heavy and light chain variable regions for CR3009, were synthesised (Genewiz) based on the GenBank sequences with regions of overlap to restriction digested human IgG1 vectors for assembly cloning (NEB) to produce plasmids: CR3009HC and CR3001KC. N-protein specific mAb CR3009 was produced by co-transfecting Expi293F cells (Life Technologies) in suspension growing at 37°C in 8% CO₂ atmosphere in FreeStyle 293T medium (Life Technologies) with the plasmids. The supernatants were harvested 6–8 days post-transfection. as per the original study describing this mAb (van den Brink et al. 2005). The CR3009 Mab were purified by affinity chromatography using a 5 ml Protein G column (Cytiva) attached to an AKTA Pure system. Upon loading, the column was washed with PBS and bound Mabs eluted with 0.1 M glycine pH 2.2 and immediately neutralised with 1 M Tris, pH 8.0. The mAb-containing fractions were pooled and subjected to Size Exclusion Chromatography using a Superdex 200 16/600 prep grade column. The purified mAb CR3009 was labelled with Alexa Fluor 488-NHS (Cat#1812 Jena Biosciences) according to the instructions from the manufacturer.

Heavy and light chain variable regions for CR3009, were synthesized (Genewiz) based on the GenBank sequences with regions of overlap to restriction digested human IgG1 vectors for assembly cloning (NEB) to produce plasmids: CR3009HC and CR3001KC. N protein specific mAb CR3009 was produced by co-transfecting Expi293F cells (Life Technologies) in suspension growing at 37°C in 8% CO₂ atmosphere in FreeStyle 293T medium (Life Technologies) with the plasmids. The supernatants were harvested 6–8 days post-transfection. as per the original study describing this mAb [77 (link)]. The CR3009 Mab were purified by affinity chromatography using a 5 ml Protein G column (Cytiva) attached to an AKTA Pure system. Upon loading, the column was washed with PBS and bound mAbs were eluted with 0.1 M glycine pH 2.2 and immediately neutralized with 1 M Tris, pH 8.0. The mAb-containing fractions were pooled and subjected to size exclusion chromatography using a Superdex200 16/600 prep grade column. The purified mAb CR3009 was labelled with Alexa Fluor 488-NHS (Cat#1812 Jena Biosciences) according to the instructions from the manufacturer.

Total IgG was affinity-purified on a protein g column (Cytiva; Uppsala, Sweden. The sera were loaded on the column and washed with PBS, and bound antibodies were eluted with 0.2 M glycin-HCl pH 2.2, neutralized to pH 7, and dialyzed against PBS. Total IgG concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific; Rockford, IL, USA). IgGs were filtered using a 0.22 µm syringe filter before their use for the in vitro or in vivo experiments.

The Sema3G expression vector has been described previously [12 (link)]. The vector was transfected into 293T cells using Lipofectamine LTX (Thermo Fisher Scientific). Cells were cultured in DMEM supplemented with ultra-low IgG FBS (Gibco). The culture supernatant was collected and dialyzed using Amicon Ultra 100 K (Merck). The dialyzed supernatant was further purified with a Protein G column (Cytiva), and the buffer was exchanged with PBS. Sema3G production was confirmed by Coomassie blue staining and western blotting.