Chinese hamster ovary cells were infected with 50 pfu/cell of RAd Fc, RAd SNC-Fc, or RAd EC4-Fc or subjected to AMAXA technology–mediated transfection of plasmids for mutated versions of SNC-Fc. The conditioned media was collected at 66 and 138 hours after infection/transfection. The conditioned media was pooled, and protein purification was achieved with protein A columns (Amersham Biosciences). The protein concentration was determined using the Bradford Protein assay (Sigma) and compared using western blotting.
Protein a column
Manufactured by Cytiva
Protein A columns are laboratory equipment used for the purification and isolation of antibodies or antibody fragments from complex samples, such as cell culture supernatants or serum. The columns contain an immobilized form of Protein A, a bacterial protein that has a high affinity for the Fc region of antibodies. This allows the antibodies to be selectively captured and separated from other proteins in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay, by Merck Group (3 mentions)
Expi293 expression system, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Euroclone (1 mentions)
Dulbecco modified eagle medium (dmem), by Euroclone (1 mentions)
Akta pure, by Cytiva (1 mentions)
Ultra low igg fcs, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Pierce chromogenic endotoxin quant kit, by Thermo Fisher Scientific (1 mentions)
Expicho cells, by Thermo Fisher Scientific (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Dynamis medium, by Thermo Fisher Scientific (1 mentions)
Hitrap desalting column, by Cytiva (1 mentions)
Superdex 200 column, by Cytiva (1 mentions)
16 protocols using protein a column
1
Purification and Characterization of Recombinant Fc-Fusion Proteins
2
Purification of RAd-Fc Fusion Proteins
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CHO cells were infected with 50 pfu/cell of RAd Fc or RAd SNC-Fc, or alternatively transfected using Amaxa nucleofection with the mutated SNC-Fc plasmids following the manufacturer's instructions. The conditioned media was collected at 66 and 138 h after infection. Conditioned media was pooled and purification achieved with protein A columns (Amersham Biosciences). Protein concentrations were determined using the Bradford Protein assay (Sigma).
3
Generation of TOSV-specific antibodies
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PBMCs and plasma samples were obtained from a patient with meningitis, infected by TOSV, during the acute phase and one year after the infection. At the Institute for Research in Biomedicine (Bellinzona Switzerland), frozen PBMCs were thawed and immortalized using EBV in the presence of CpG, as previously described [26 (link)]. Briefly, IgG+ memory B cells were isolated using magnetic cell separators (MACS) and negative and positive selection kits (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, memory B cells were seeded at 10 or 50 cells per well in 96 U-bottom microplates (Greiner, ThermoFisher Scientific) in complete medium containing 2.5 μg/mL CpG 2006, in the presence of EBV (30% supernatant of B95-8 cells) and irradiated allogeneic mononuclear cells (50,000 per well). After 2 weeks, the culture supernatants were screened for specific antibodies by Intellicyt Cytometer against TOSV-infected VERO cells. Specific TOSV antibodies were purified from culture supernatants by affinity chromatography on protein A columns (Amersham) and stored at −20 °C for further testing.
4
Protein Expression and Purification
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CHO cells were transduced with 50 pfu/cell of RAd Fc or RAd EC4-Fc as described previously14 (link). The conditioned media was collected at 66 and 138 hours after transduction/transfection. The conditioned media was pooled and protein purification was achieved with protein A columns (Amersham Biosciences). The protein concentration was determined using the Bradford Protein assay (Sigma) and compared by Western blotting.
5
Monoclonal Antibody Production Process
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Antibody heavy chain and light chain variable regions were cloned into human immunoglobulin G (IgG), human IgK, and human IgL backbone constructs. These were transiently transfected and expressed using the Expi293 Expression System (Thermo Fisher Scientific). After 6 days, culture supernatants were harvested, spun down, and sterile-filtered. mAbs were purified from supernatant using Protein A columns (Cytiva) and eluted under sterile conditions. Purified mAb used for in vivo experiments was then confirmed to be endotoxin-free (Pierce).
6
Chimeric Antibody Characterization Protocol
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TC6, TF9, and BH6 were produced as chimeric antibodies with a human IgG1 Fc domain by Evitria. Antibodies purified on protein A columns (Cytiva) and were tested in ELISA against plate bound antigen, with detection using goat anti-human IgG (H+L) (SeraCare). Cross-reactivity against mouse B7-H3 was tested using flow cytometry against the mouse cell line 3T3/NA1.
7
Monoclonal Antibody Production Process
8
Monoclonal Antibody Production from Hybridoma Cells
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The mAb-hERG1 (MCK Therapeutics; Florence, Italy; patent number IT1367861) was produced by secretion from an hybridoma cell line (named A7) obtained from mice cell fusion and able to secrete the monoclonal antibody (Guasti et al., 2008 (link); Lastraioli et al., 2020 (link); Lottini et al., 2021 (link)). Hybridoma cells are routinely maintained in DMEM (Euroclone) supplemented with Glutamine 10 mM and 5% Fetal Clone serum (Euroclone). Supernatant is harvested and purified by affinity chromatography using AKTA Pure (Cytiva) mounted with Protein A columns (Cytiva).
9
Rabbit Immunization for Anti-Malarial IgG
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New Zealand rabbits were immunized and bled at Kaneka Eurogentec S.A. (Belgium). The animals (n = 2) were given three subcutaneous immunizations (30 μg antigen per dose) in intervals of 4 weeks. Blood was collected before each immunization and 9 days after the final injection. Total serum IgG was purified from rabbit sera using protein A columns (Cytiva) and assessed for activity in a single-cycle in vitro GIA with the Pf 3D7 strain.
10
Engineered Antibody Production
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All antibodies (MOPC21 isotype control, nivolumab, OX108) were designed as mIgG1 isotype with a D265A mutation to reduce Fc gamma receptor (FcγR) binding [25 (link)]. The heavy chain constant region included mutations that improve Protein A binding [26 (link)] without affecting FcRn binding (personal communication by Absolute Antibodies Ltd.). Heavy and light chains were ligated into pHR-SIN-IRESEm. HEK293T cells were co-transfected with the respective pHR plasmid as well as pmDG and p89.1 plasmids for lentivirus production. Subsequently, CHO-K1 cells were transduced with equal amounts of viruses containing the respective heavy and light chains. Once cells expanded, the medium was switched to 1% ultra-low IgG FCS (ThermoFisher) with additional supplementation of 1 × non-essential amino acids (ThermoFisher). Supernatants were harvested every week and affinity-purified using Protein A columns (Cytiva). All antibodies were size-excluded using a ProteoSEC 16/60 6-600 HR SEC column on an endotoxin-free ÄKTA start machine. All antibodies were confirmed to be endotoxin-free using the Pierce Chromogenic Endotoxin Quant Kit (ThermoFisher).
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