Hitrap protein g hp column

BALB/c mice were immunized intraperitoneally on days 0, 7, 14, and 21 with 25 μg of purified recombinant proteins, emulsified in Sigma Adjuvant System (Sigma, Cat. # S6322). Mice were boosted on day 28 intravenously with 10 μg of the protein. Three days later, mice were euthanized and the spleen cells used for fusion to NS0 myeloma cells. Polyethylene glycol was used as the fusing agent and the cells were incubated for 16 h. Subsequently, HAT (hypoxanthine, aminopterin, and thymidine) (Sigma, Cat. # H0262) was added to the medium^{69 (link)}. The hybridomas were cloned by limiting dilution and the supernatants were screened by western blotting and indirect immunofluorescence with G. lamblia trophozoites (mAb 7F5, anti-VSP1267) or by western blotting and enzyme-linked immunosorbent assay (ELISA; mAb 15E4, anti-HA H5N1). MAbs were purified from supernatants screened using the ÄKTA® pure chromatography apparatus (HiTrapTM Protein G HP column, GE Cat. # 17-0405-03).

Antibody responses against HA of UDL1/08 were induced in two adult Biozzi ABH mice (Harlan^®). Vaccination consisted of an initial subcutaneous (SC) inoculation of 5000 pfu in 100 μL of recombinant adenovirus H9HA (rAd-H9HA). Twenty one days later, each mouse was boosted with 5000pfu (in 100 μL) of MVA H9HA (rMVA-H9HA) via the SC route. Forty eight days after the rMVA-H9HA boost, each mouse was additionally boosted with 5 μg (in 100 μL) of recombinant H9HA purified protein (produced in S2 cells) via the intravenous route. Four days later mice were humanely euthanized and splenocytes were harvested for fusion with myeloma cells. Hybridoma cells expressing mAbs against H9HA were selected by ELISA against purified UDL1/08 virus adopting a standard mAb screening method51 (link).
Hybridoma stocks were grown in suspension using IgG-depleted serum using the miniPERM Bioreactor (Sarstedt). Monoclonal antibodies in the miniPERM supernatant were then purified by affinity purification using a HiTrap Protein G HP column (GE healthcare) and dialysed into PBS.

The mouse pan-flavivirus antibody 4G2 expressed by hybridoma cells was purified from cell supernatants using a HiTrap Protein G HP column from GE Healthcare (Chicago, IL, USA) according to the manufacturer’s recommendations.

The hybridoma lines were cultured in Hi‐Growth Hybridoma Media (RPMI 1640 supplemented with 10% FBS, 10% NCTC‐109, 20 mmol/L HEPES, 2 mmol/L L‐glutamine, 1X NEAA, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 1X Hybridoma Fusion and Cloning Supplement (Roche)). Supernatants were collected and antibodies were purified using a HiTrap Protein G HP column (GE Healthcare) with a running buffer of 20 mmol/L sodium phosphate, pH 7.0, and eluted with 100 mmol/L glycine‐HCl, pH 2.7 into faction tubes containing 1 M Tris, pH 9.0. Antibody containing fractions were pooled, dialysed with 1x DPBS (Life Technologies), concentrated with 30 kDa MWCO filters and stored in 1x DPBS, 10% glycerol, 0.02% NaN3. All mAbs were purified by SEC to remove protein aggregates and immediately used for the assays, in order to avoid potential artefacts due to avidity effect.

The parental hybridoma clone secreting PRRSV-specific mAb-PN9cx3 was generated as previously described [27 ]. Briefly, mice were immunized with SF9 cells infected with recombinant baculovirus expressing GP3 protein of strain VR2385 and then subjected to hybridoma fusion based on standard fusion protocols. The supernatants of the surviving hybridomas were screened using immunofluorescence assays (IFAs) to detect the immunofluorescence of PRRSV VR2385-infected CRL-11171 cells (a parallel subcloned cell line derived from MA104). Parental hybridoma cell clones were propagated via three additional rounds of subcloning to obtain the mAb-PN9cx3-producing clone. mAb-PN9cx3 was purified from mouse ascites using a HiTrap Protein G HP column (GE Healthcare) in accordance with the manufacturer’s instructions. Mouse ascites containing mAb-PN9cx3 were generated by Genscript Co., Ltd., in BALB/c mice (Nanjing, Jiangsu, China). The purified mAb was eluted with 0.1 M glycine (pH 2.7), dialyzed against phosphate-buffered saline (PBS) overnight at 4 °C, concentrated using 100-kDa cutoff ultrafiltration centrifugal tubes (EMD Millipore) and quantified using a BCA protein quantification kit (Thermo Fisher Scientific). The previously described mAb-2G8 (IgG) against HEV-ORF2 protein was used as an isotype control and was purified using the same methods used to purify mAb-PN9cx3 [28 (link)].

Mouse GD2-binding mAbs, 14G2a (IgG2a) were purified from 14G2a hybridoma culture supernatants using the HiTrap Protein G HP column (17-0404-01, GE Healthcare Bio-Sciences AB) according to the manufacturer’s protocol. Antibodies were dialysed using D-Tube ^TM Dialyzer Midi tubes (71507-3, Millipore) against 4 liters of PBS (phosphate-buffered saline, pH 7.3–7.5) for 24 h at 4 °C. Protein concentration was measured using the BCA assay (B9643, C2284, Sigma-Aldrich) according to the manufacturer’s protocol.