Pcdna3.4 vector

Selected antibody sequences were purchased as gBlocks gene fragments (Integrated DNA Technologies) and cloned into a customized pcDNA3.4 vector (Invitrogen) containing human IgG1 or IgA1 Fc regions. VH and VL plasmids were transfected into 30 mL cultures of Expi293F cells (Invitrogen) at a 1:2 ratio and incubated at 37˚C and 8% CO₂ for 7 days. The supernatant containing secreted antibodies was collected following centrifugation (1000 g for 10 min at 4˚C), neutralized, and filtered. Antibodies were isolated using Protein G Plus agarose (IgG; Pierce Thermo Fisher Scientific) or Peptide M agarose (IgA; Invitrogen) affinity chromatography, washed with 20 column volumes of PBS, eluted with 100 mM glycine-HCl pH 2.7, and immediately neutralized with 1 M Tris-HCl pH 8.0. The antibodies were then concentrated and buffer exchanged into PBS using 10,000 MWCO Vivaspin centrifugal spin columns (Sartorius).

A codon-optimized sequence of full length ETV1 was cloned into a pcDNA3.4 vector (Invitrogen). Synthesized ETV1 sequence included at C-terminus a FLAG tag sequence and a streptavidin binding peptide sequence (SBP tag). The vector was transfected into HEK293F (Invitrogene) cells adapted to grow in suspension to enable the up scaling of protein production. After 72 hours the cells were harvested and lysed in 1xRIPA buffer supplemented with 2× complete protease inhibitors (Roche). The lysates were cleared by centrifugation at 15,000 rpm for 20 min at 4°C and filtered trough a 0.2 μm filter. The ETV1 protein was bound to a streptavidin column via SBP tag and eluted in 2 mM biotin in PBS buffer.

Selection of antibody sequences for recombinant expression was based on the combination of V_H:V_L-paired databases and proteomics data. First, we identified antibody clonotypes identified in the proteomics analysis and searched for the same clonotype in the V_H:V_L-paired database. Full-length heavy and light chain sequences were then determined from the paired sequencing database.
These genes were purchased as gBlocks (Integrated DNA Technologies) and cloned into the pcDNA3.4 vector (Invitrogen). Heavy and light chain plasmids for each monoclonal antibody were transfected into Expi293 cells (Invitrogen) at a 1:3 ratio. After incubating for 5 days at 37°C with 8% CO₂, the supernatant containing secreted antibodies was collected by centrifugation at 500×g for 15 min at RT. Supernatant was passed over a column with 0.5 ml Protein A agarose resin (Thermo Scientific) three times to ensure efficient capture. After washing the column with 20 cv of PBS, antibodies were eluted with 3 ml 100 mM glycine-HCl, pH 2.7 and immediately neutralized with 1 ml 1 M Tris-HCl, pH 8.0. Antibodies were buffer-exchanged into PBS utilizing Amicon Ultra-30 centrifugal spin columns (Millipore).