Protein g affinity chromatography

BALB/c and C57BL/6 mice were immunized with chemically modified (*) citrulline peptides (PIECit*TYLK-NH₂ and YAGCit*LLTK-NH2), coupled to either keyhole limpet hemocyanin (KLH) or ovalbumin. Different citrulline-containing peptides were created in the host laboratory via solid phase peptide synthesis, as described previously (20 (link)). Hybridoma cell lines were created using the ClonaCell®-HY kit (Stemcell Technologies) according to the manufacturer’s instructions. SP2/0 mouse myeloma cells were fused with purified splenocytes of immunized mice. Stable hybridomas were screened and selected on the basis of antibody reactivity to different synthetic citrullinated and control peptides that were coated on the plates at 100 ng/ml with the use of indirect ELISAs (20 (link)). Further validation was performed by Western Blot analysis and the use of synthetic citrullinated and non-citrullinated CXCL8 isoforms (21 (link)). Cell culture supernatants were filtered and antibodies were purified on Protein G affinity chromatography (GE Healthcare).

Each New Zealand white rabbit (n = 3) was subcutaneously immunized with 100 μg of each purified recombinant protein emulsified with and without Freund’s adjuvant as described previously [24 (link)]. Immunoglobulin G was purified by protein G affinity chromatography (GE Healthcare), and the concentration was determined using BCA protein assay kit (Beyotime).

The mouse antibody (Ab) variable regions of heavy and light chains were amplified with PCR using previously described primers and conditions (51 (link)). PCR products were purified and cloned into an expression vector encoding the mouse heavy or light chain constant regions using ligation-independent cloning (51 (link)). Ab variable regions were sequenced by Sanger sequencing, and germ lines were determined using IMGT, the international ImMunoGeneTics information system (http://www.imgt.org) (52 (link)).
Ab heavy and light chain plasmids were cotransfected at a 1:1 ratio into human embryonic kidney (HEK) 293F cells (Thermo Fisher Scientific) using PEI Max 40K (linear polyethylenimine hydrochloride; Polysciences, Inc.). Ab supernatants were harvested 5 days following transfection and purified using protein G affinity chromatography following the manufacturer’s protocol (GE Healthcare).

The coding sequences for variable domains of antibody 492.1 were fused to human antibody constant regions [72 (link)]. Mutations were introduced by site-directed mutagenesis into the resulting hybrid antibody expression plasmids according to published procedures [73 (link)]. Plasmids were transfected into suspension-adapted suspension-HEK 293F cells. The day before transfection, cells were split to 0.7 x 10⁶ cells/ml. For parallel expression of the parent hybrid antibody and the 20 variants, transfections were performed using 0.5 μg of each plasmid (heavy and light Ab chains) mixed with 3 μg PEI Max reagent (Polysciences Inc.) and incubated 20 min in 24-well tissue culture trays prior to addition of 1 ml cells per well. Plates were then agitated vigorously in a tissue culture incubator/shaker to prevent cell settling. After 4 days, cultures were transferred to microfuge tubes, and cells were pelleted by centrifugation at 500 x g for 10 min. Supernatants were transferred to fresh microfuge tubes, from which aliquots were taken for quantification of antibody expression and activity. For purification of selected Ab designs, transfections were done in 40 ml volumes, and plasmid and PEI Max amounts were scaled up accordingly. Cultures were grown for 6 days, and Ab was purified from the supernatant by protein G affinity chromatography (GE Healthcare).

Hybridoma supernatants were incubated with each of the anti-isotype (anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3, anti-IgA, and anti-IgM antibodies) previously coated at MaxiSorp microplates followed by incubation with horseradish peroxidase-conjugated rabbit anti-mouse-IgG + A + M (1:1000) (Zymed, San Francisco, CA, USA). The supernatants from selected clones were filtered (0.45 μm) and purified by protein G affinity chromatography (GE Healthcare Life Science, Freiburg, Germany). mAb purity was observed in a 12% SDS-PAGE staining with Coomassie blue R-250. The detection limit was established using ZIKV-NS1 concentrations from 1 μg/mL to 0.6 ng/mL coated on microplates, followed by incubation with 10 μg/mL or 2.5 μg/mL of different anti-ZIKV-NS1 mAbs and with goat anti-IgG mouse conjugated with horseradish peroxidase (HRP) (1:5000) (Invitrogen, Carlsbad, CA, USA).