The modified high-affinity xfR5 mAb was produced and isolated from a hybridoma cell line, i.e., XF27/28/CCR5 43E2-AA (PTA-4054; ATCC repository) [32 ], by following the published method with modifications as described below [37 (link)]. Briefly, XF-CCR5 28/27 43E2AA hybridoma cells were seeded (at 106/mL concentration) and maintained in antibody production inducing media, i.e., RPMI medium with 1 × AA, for several days until 50% cells were found to be compromised (cell death). The supernatant with soluble xfR5 mAb was harvested by pelleting out dead cells and debris. The soluble xfR5 mAb from the supernatant was isolated using HiTrap™ Protein-A HP prepacked column (GE Healthcare; Chicago, IL, USA) following standard manufacturer’s protocol. The purity and concentration of the xfR5 mAbs were determined by the SDS-PAGE method and BCA assay using Pierce™ BCA Protein Assay Kit, following manufacturers’ protocol. The xfR5 binding was evaluated based on the standard curve (linear regression analysis) from a known concentration of IgG4 isotype control mAb, as xfR5 is recombinant human CCR5 mAb with IgG4 isotype backbone [32 ].
Hitrap protein a hp prepacked column
Manufactured by GE Healthcare
Sourced in United States
The HiTrap™ Protein-A HP prepacked column is a ready-to-use chromatography column for the purification of antibodies. It is prepacked with Protein A agarose, a ligand with high affinity for the Fc region of immunoglobulins. The column is designed for fast and efficient antibody capture and purification.
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2 protocols using hitrap protein a hp prepacked column
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Production and Purification of Modified xfR5 mAb
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Production and Purification of xfR5 mAb
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The modi ed high a nity xfR5 mAb were produced and isolated from a Hybridoma cell line, i.e., XF27/28/CCR5 43E2-AA (PTA-4054; ATCC repository) [60], by following published method with modi cations as described below [62] . Brie y, XF-CCR5 28/27 43E2AA hybridoma cells were seeded (at 10 6 /mL concentration) and maintained in antibody production inducing media, i.e., RPMI medium with 1×AA, for several days until 50% cells were found to be compromised (cell death). The supernatant with soluble xfR5 mAb was harvested by pelleting out dead cells and debris. The soluble xfR5 mAb from the supernatant was isolated using HiTrap™ Protein-A HP prepacked column (GE Healthcare; NJ, USA) following standard manufacturer's protocol. The purity and concentration of the xfR5 mAbs were determined respectively by the SDS-PAGE method and BCA assay using Pierce™ BCA Protein Assay Kit, following manufacturers' protocol. The xfR5 binding was evaluated based on the standard curve (linear regression analysis) from a known concentration of IgG4 isotype control mAb, as xfR5 is recombinant human CCR5 mAb with IgG4 isotype backbone [60] .
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