Hitrap protein g hp

Canine adenovirus 2 was propagated and purified, then used to produce mAbs as previously described (Huang et al., 2007 (link); Yin et al., 2012 (link)). Briefly, five 6-week-old female BALB/c mice were immunized with 60 μg purified CAdV-2 emulsified with complete Freund’s adjuvant (Sigma-Aldrich). Two booster immunizations containing purified CAdV-2 and equal volume of incomplete Freund’s adjuvant (Sigma-Aldrich) were performed at 2-week intervals. Three days after the final booster, spleens were removed and splenocytes were fused with SP2/0 myeloma cells. Hybridoma cell lines secreting antibodies against CAdV-2 were screened and subcloned at least three times by limiting-dilution (Greene et al., 1980 (link)). Hybridoma culture supernatants were screened for antibodies using indirect ELISA. Antibodies that bound to the CAdV-2 virus but failed to bind MDCK cells were considered CAdV-2-positive. The stable cells were injected into the abdominal cavities of BALB/c mice, which had been pretreated with liquid paraffin. Approximately 1 week later, the mAbs were harvested and purified from the seroperitoneum using an antibody purification kit (HiTrap Protein G HP, GE Healthcare, Milwaukee, WI, United States) per the manufacturer’s instructions.

Immunoglobulins G were isolated from individual serum samples of schizophrenia patients and healthy controls by affinity chromatography on an ÄKTA Pure 25 chromatograph (GE Healthcare Bio-Sciences, Uppsala, Sweden). Serum after 3-fold dilution with Tris-buffered saline (TBS; 0.05 M Tris-HCl pH 7.5 and 0.15 M NaCl) was applied to a Protein G Sepharose column (HiTrap™ Protein G HP, GE Healthcare). After the sample loading, the column was washed to zero optical density at 280 nm (A₂₈₀) with TBS. At the first step, the elution of lipids and nonspecifically adsorbed proteins was performed with TBS-Tween buffer (1% Triton X-100, 0.05 M Tris-HCl pH 7.5, and 0.3 M NaCl). IgGs were eluted with 0.1 M glycine-HCl buffer (pH 2.6). The eluate was fractionated and neutralized immediately with 1 M Tris-HCl buffer (pH 8.8). Between the stages of antibody purification, the columns were washed with TBS until A₂₈₀ of the eluate reached zero [20 (link)]. Then, the homogeneity of IgGs was tested by SDS-PAGE analysis in a 4–18% gradient gel.

A recombinant sPTPRG derivative consisting of the extracellular domain without transmembrane segment and cytoplasmic tail, was expressed in HEK 293F cells. To obtain the PTPx-FcIgG3 construct, the cDNA fragment corresponding to extracellular domains of PTPRG 1 through 736 was cloned into pCEP4 plasmid carrying a mouse IgG3 domain between KpnI and BamHI and was inserted in frame between Bcl I (inside IgG3 domain) and Kpl I. Transfected HEK 293F cells were cultured in RPMI 1640 containing 10% heat-inactivated Fetal Calf Serum (FCS), 4mM glutamine, and 0,5 mg/mL of hygromycin (Invitrogen Milan, Italy) as selective agent or cultured in serum free medium CD293 (Gibco, Milan, Italy) with 50 μM β-mercaptoethanol and 0,5 mg/mL of hygromycin (Invitrogen, Milan, Italy). All the cultured cells were grown at 37°C, in 5% CO₂. The recombinant ECD-PTPRG-Fc fusion protein was purified from serum free medium cultured cell by affinity chromatography using protein-G Sepharose according to the manufactory protocol of HiTrap Protein G HP, 1 ml (GE Healthcare, Milan, Italy). The yield was around 0,2 mg/ml for >80% of purity.

The immunodominant peptide (VMRIVRALPE) was chemically synthesized and coupled to Keyhole limpet hemocyanin (KLH) using standard methods at the Facility for Biotechnology Resources, CBER, FDA^{52 (link)}. Rabbits were immunized with 125 μg per dose of either the KLH-peptide or the purified recombinant Tc_5171 antigen, with three antigen boosts at 21-day intervals to generate polyclonal sera. Antibodies against the VMRIVRALPE peptide were affinity purified from the serum of the immunized rabbit using the SulfoLink Immobilization Kit for Peptides from Thermo Fisher. Briefly, the VMRIVRALPE peptide, synthesized with an N-terminal cysteine was covalently coupled to the iodoacetyl group on agarose beads. Peptide coupled beads were incubated with 1:2 dilution of rabbit serum in PBS. Peptide bound antibodies were eluted using 0.1 M Glycine HCl and neutralized using 1 M Tris, HCl pH 9.0. Polyclonal IgG antibodies generated against the full-length recombinant Tc_5171 antigen were purified using a HiTrap Protein G HP antibody purification column from GE Lifesciences using manufacturer recommended protocol. Purified antibodies were concentrated and dialyzed in HBS.

Culture supernatants were filtered through a 0.22 μm filter (Millipore, USA), and then Hitrap Protein G HP (1 mL, GE Healthcare, USA) was used to purify IgG according to a previously published procedure (9 (link)). Briefly, culture samples stimulated with various cytokines were pumped into the affinity column, and unbound proteins were removed using five column volumes of binding buffer (0.02 MTris, pH=7.2). Bound IgG was eluted with elution buffer (0.1 M glycine, pH=2.7), and the eluate was neutralized to pH 7.2 immediately. Then, IgG samples were desalted and exchanged in PBS buffer using a PD-10 Desalting Column (GE Healthcare, USA) according to the manufacturer’s instructions, and the IgG solutions were concentrated using a 10-kD ultrafiltration tube (Millipore, USA). The purified IgG samples were stored at -80°C until further use.