The selected hybridoma cell clones were incubated in serum-free medium, Hybridoma-SFM (Thermo Fisher Scientific, MA, USA), and the medium from the expanded culture of the selected clones was centrifuged to exclude cell debris. Monoclonal antibodies in the semi-purified medium bound to rProtein A Sepharose Fast Flow resin (GE Healthcare, Buckinghamshire, England) overnight using a circulation pump. Then, the columns were washed with 10x the volume of PBS, and the bound monoclonal antibodies were eluted with 0.1 M sodium citrate (pH4.0), neutralized with 1.0 M Tris-HCl (pH 9.0), and subjected to dialysis with PBS using the recommended protocol.
Rprotein a sepharose fast flow resin
Manufactured by GE Healthcare
Sourced in United Kingdom
RProtein A Sepharose Fast Flow resin is a chromatography medium used for the purification of antibodies and Fc-containing proteins. It consists of highly cross-linked agarose beads with covalently coupled recombinant Protein A. The resin provides a high binding capacity and fast flow rates for efficient antibody capture and purification.
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Lab products found in correlation
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (4 mentions)
Amicon centrifugal filter, by Merck Group (4 mentions)
Kta fplc system, by GE Healthcare (2 mentions)
Talon metal affinity resin, by Takara Bio (2 mentions)
Optipro serum free medium, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Protein g chromatography cartridge, by Thermo Fisher Scientific (2 mentions)
Hybridoma sfm, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Sterile pbs, by Corning (1 mentions)
Hiload 16 600 superdex 200, by GE Healthcare (1 mentions)
Lal assay, by Lonza (1 mentions)
Steriflip filter unit, by Merck Group (1 mentions)
Talon resin, by Takara Bio (1 mentions)
Freestyle medium, by Thermo Fisher Scientific (1 mentions)
Cho k1, by American Type Culture Collection (1 mentions)
Biorad protein concentration reagent, by Bio-Rad (1 mentions)
Capto, by Cytiva (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qcl 1000 chromogenic lal assay, by Lonza (1 mentions)
12 protocols using rprotein a sepharose fast flow resin
1
Purification of Monoclonal Antibodies
2
Purification and Characterization of Cytokine-Fusion Proteins
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Suspension HEK293 cells were transfected with sterile-filtered plasmid DNA using polyethylenimine in OptiPro serum-free medium (Thermo Fisher). TA99 was purified using rProtein A Sepharose Fast Flow resin (GE Healthcare) as previously described40 (link). His-tagged proteins were isolated from HEK293 supernatant using TALON Metal Affinity Resin (Takara Bio Inc.). Some cytokine-fusion proteins were then further purified by size exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column on an ÄKTA FPLC system (GE Healthcare) that had been pretreated overnight with 1 M NaOH to remove endotoxin and subsequently equilibrated in sterile PBS (Corning). After purification, all proteins were buffer exchanged into sterile PBS (Corning), 0.2 μm sterile-filtered (Pall Corporation), and confirmed to contain minimal endotoxin (<0.1 EU per injection) using a chromogenic LAL assay (Lonza). To confirm their molecular weights, proteins were run alongside a Novex Prestained Sharp Protein Ladder on a 4–12% NuPAGE Bis-Tris protein gel (Life Technologies) with 1% MES running buffer. All proteins were stored at −80 °C, but before therapeutic injection or in vitro assessment, cytokine fusion proteins were thawed on ice.
3
Monoclonal Antibody Production Protocol
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Monoclonal antibodies were obtained as previously described [9 (link)]. Briefly, the sequences of CIS43 [7 (link)], L9 [9 (link)], 317 [6 (link)], VRC01 [25 (link)], and control antibody [12 (link)] were retrieved from PDB or GenBank and cloned into the pVRC8400 huIgG1, pVRC8400 huIgK, or SBShuLambda expression vectors (GenScript) containing the relevant constant region. Matched heavy and light chain constructs were co-transfected into Expi293 cells using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific) and cultures were incubated at 37°C, 8% CO2 for 6 days. Supernatants were harvested and purified using rProtein A Sepharose Fast Flow resin (GE Healthcare) and buffer exchanged with 1X PBS (pH 7.4) before being concentrated using Amicon Centrifugal Filters (Millipore) and sterile filtered through a 0.2 μm Steriflip filter units (Millipore). Purified mAbs were diluted to appropriate concentrations in sterile 1X PBS and concentrations were confirmed using a Nanodrop spectrophotometer.
4
Production and Purification of Murine PD-1 Protein
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Proteins were produced in-house using suspension HEK293-F (Life Technologies, R79007), which were cultured in Freestyle medium (Life Technologies). HEK293 cells were transfected with sterile-filtered plasmid DNA using polyethylenimine in OptiPro serum-free medium (Thermo Fisher). The murine PD-1 ectodomain monoFc protein fusion and monovalent antibodies were HIS-tagged and purified by gravity column using TALON resin (Takara Bio Inc.). All bivalent antibodies were purified by gravity column using rProtein A Sepharose Fast Flow resin (GE Healthcare). All proteins were characterized by Coomassie-stained SDS NuPAGE Bis-Tris protein gels (Thermo Fisher Scientific). Proteins were further purified by size exclusion chromatography, if aggregation was detected on the protein gel, using a HiLoad 16/600 Superdex 200 pg column (Millipore Sigma) on an ÄKTA FPLC system (GE Healthcare). After purification, all proteins were buffer exchanged into sterile phosphate-buffered saline (PBS) (Corning), 0.2 μm sterile-filtered (Pall Corporation), and confirmed to contain minimal endotoxin (<0.1 EU per injection) using a chromogenic LAL assay (Lonza). All proteins were flash-frozen by liquid nitrogen, stored at −80°C and thawed on ice before use.
5
Characterization and Biotinylation of hJAA-F11 H2aL2a
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The characterization of the hJAA-F11 H2aL2a has been detailed elsewhere [37] (link). Briefly, two vectors, encoding the heavy and light chains of H2aL2a, were transfected into the Chinese hamster ovary cell line CHO-K1 (ATCC, Manassas, VA). Stable cell lines expressing hJAA-F11 H2aL2a were selected using G418 and histidinol and confirmed to bind to immobilized TF-Ag-α linked to BSA in an enzyme immunoassay [17] (link), [39] (link). hJAA-F11 H2aL2a was then purified using rProtein A Sepharose Fast Flow resin (GE Healthcare, Pittsburgh, PA), followed by Capto S ImpAct resin (GE Healthcare, Pittsburgh, PA).
Purified hJAA-F11 H2aL2a was dialyzed in 1× PBS and biotinylated using an ImmunoProbe Biotinylation Kit (Millipore-Sigma, St. Louis, MO) according to the manufacturer’s protocol. The same protocol was used to also biotinylate a matched human IgG1 myeloma protein (Fitzgerald, Acton, MA). The ability of biotinylated hJAA-F11 H2aL2a to recognize TF-Ag-BSA was confirmed using ELISA probed with streptavidin linked to alkaline phosphatase. Antibody purity was determined using SDS-PAGE to confirm that there was no change in antibody structure such as cross-linking or degradation following modification. Concentrations were determined using A280 absorbance as well as a BioRad protein concentration reagent (BioRad, Hercules, CA).
Purified hJAA-F11 H2aL2a was dialyzed in 1× PBS and biotinylated using an ImmunoProbe Biotinylation Kit (Millipore-Sigma, St. Louis, MO) according to the manufacturer’s protocol. The same protocol was used to also biotinylate a matched human IgG1 myeloma protein (Fitzgerald, Acton, MA). The ability of biotinylated hJAA-F11 H2aL2a to recognize TF-Ag-BSA was confirmed using ELISA probed with streptavidin linked to alkaline phosphatase. Antibody purity was determined using SDS-PAGE to confirm that there was no change in antibody structure such as cross-linking or degradation following modification. Concentrations were determined using A280 absorbance as well as a BioRad protein concentration reagent (BioRad, Hercules, CA).
6
Purification of anti-PfCSP human monoclonal antibodies
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The isolation of PfCSP hmAbs used in this study was previously reported.8 (link)–12 (link) All hmAbs were expressed in IgG1 heavy and light chain plasmids using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) and incubated at 37°C, 8% CO2 for 6 days. Supernatants were harvested and purified using rProtein A Sepharose Fast Flow resin (GE Healthcare) or protein G chromatography cartridge (Thermo Fisher Scientific). Buffer was exchanged with 1X PBS before being concentrated using Amicon Centrifugal Filters (Millipore). The concentration of purified antibodies was determined using the Nanodrop spectrophotometer (Thermo Fisher Scientific).
7
Production and Purification of Therapeutic Proteins
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Therapeutic proteins and vaccines were produced as described [20 (link)]. Total RNA was isolated from B16F10 cells with RNeasy Mini Kit (Qiagen) and reverse transcribed with SuperScript III RT and random primers (Invitrogen) to synthesize B16F10 first-strand cDNA. eMLV env and gag sequences were amplified from B16F10 first-strand cDNA. eMLV env monomeric gp70 with a C-terminal His tag was cloned into the gWIZ vector (Gelantis) by Gibson assembly and produced by transiently transfecting HEK293F cells with the plasmid and polyethylenimine. The RBD was cloned by site-directed mutagenesis (NEB) and produced as gp70. Gag was expressed as a SUMO fusion using the pE-SUMOpro vector (LifeSensors) in Rosetta-gami 2 (DE) competent cells. Heavy and light chains of anti-env Abs were separately cloned into the gWIZ vector and anti-env Abs were produced by transiently co-transfecting HEK293F cells as above. His-tagged proteins were purified using TALON Metal Affinity Resin (Takara) and Abs were purified using rProtein A Sepharose Fast Flow resin (GE Healthcare). Endotoxin levels were below 0.1 total EU/dose as measured by the QCL-1000 chromogenic LAL assay (Lonza).
8
RVFV Gn-LRP-1 Binding Assay
9
Purification of anti-PfCSP human monoclonal antibodies
10
Production and Purification of scFv Antibodies
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scFv antibody fragments were prepared according to Sokolowska-Wedzina et al. [54 (link)]. Briefly, purified pIT2 plasmids with scFv sequences were electroporated into E. coli HB2151 cells (Source BioScience). The bacteria were grown in 2× TY media supplemented with 100 μg/mL ampicillin and 0.1% glucose to OD600 = 0.8 and the production of protein was induced with 0.5 mM IPTG. Cells were cultured at 30 °C, 180 rpm, overnight and then the cultures were harvested, centrifuged twice at 4000 rcf, 4 °C for 40 min, and filtered using a Stericap PLUS bottle filter device (Merck Millipore, Darmstadt, Germany). scFv antibody fragments were purified from the supernatants by affinity chromatography using rProtein A Sepharose Fast Flow Resin (GE Healthcare), following the same protocol as described previously by our group for ECD_FGFR-Fc proteins [52 (link)]. Purified scFvs were analyzed by SDS-PAGE, Western blotting using anti-c-myc antibody, clone 9E10 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and mass spectrometry. The molecular masses of the proteins were verified by MALDI-TOF/TOF 4800 (Applied Biosystems, Foster City, CA, USA), using α-cyano-4-hydroxycinnamic acid as a matrix.
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