The soluble scFv was expressed and purified as previously described29 (link), 30 (link).The scFv-Fc variant genes were synthesized for CHO cells (Genscript), transfected into CHO-S cells and selected with G418 (Invitrogen).31 (link) High expression clones were selected for culture in Opticho serum free medium (Invitrogen),17 (link) and scFv-Fc protein purified using MabSelect affinity chromatography (GE Healthcare). After concentration with a 50,000 MWCO Vivaspin centrifuge tube (Sartorius Stedim), the scFv-Fc was tested for binding by ELISA or by flow cytometry on FACS Calibur (BD Biosciences) using peptide-loaded T2 cells.
Opticho serum free medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Opticho serum free medium is a cell culture medium designed to support the growth and maintenance of various cell types without the use of serum. It provides a defined, serum-free environment for cell culture applications.
Automatically generated - may contain errors
4 protocols using opticho serum free medium
1
Production and Purification of scFv-Fc Protein
2
Humanized 3F8 Antibody Production
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Humanized 3F8 genes were synthesized for CHO cells (Blue Heron Biotechnology or Genscript) as previously described (13 (link)). Using the bluescript vector, these heavy and light chain genes of hu3F8 were transfected into DG44 cells and selected with G418 (InVitrogen, CA, USA). Hu3F8 producer lines were cultured in Opticho serum free medium (InVitrogen) and the mature supernatant was harvested as previously described (13 (link)). Protein A affinity column was pre-equilibrated with 25 mM sodium citrate buffer with 0.15 M NaCl, pH 8.2. Bound hu3F8 was eluted with 0.1 M citric acid/sodium citrate buffer, pH 3.9 and alkalinized (1:10 v/v ratio) in 25 mM sodium citrate, pH 8.5. It was passed through a Sartobind-Q membrane and concentrated to 5–10 mg/mL in 25 mM sodium citrate, 0.15 M NaCl, pH 8.2.
3
Purification of Recombinant m708.5 IgG1 from CHO Cells
4
scFv-Fc Protein Expression and Purification
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The soluble scFv was expressed and purified as previously described.29 (link), 30 (link) The scFv-Fc variant genes were synthesized for CHO cells (Genscript, Piscataway, NJ, USA), transfected into CHO-S cells and selected with G418 (Invitrogen).31 (link) High expression clones were selected for culture in Opticho serum-free medium (Invitrogen),17 (link) and scFv-Fc protein was purified using MabSelect affinity chromatography (GE Healthcare, Piscataway, NJ, USA). After concentration with a 50 000 MWCO Vivaspin centrifuge tube (Sartorius Stedim, Goettingen, Germany), the scFv-Fc was tested for binding by enzyme-linked immunosorbent assay (ELISA) or by flow cytometry on FACS Calibur (BD Biosciences, San Jose, CA, USA) using peptide-loaded T2 cells.
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