Amicon 30 kda spin membranes

Antibodies were produced in HEK293-6E cells (National Research Council Canada) by transfection of expression plasmids for heavy and light chains by using 25 kDa branched polyethylenimine (PEI) (Sigma-Aldrich). Cells were maintained at 37°C and 6% CO2 in FreeStyle 293 Expression Medium (Thermo Fisher) and 0.2% penicillin/streptomycin (Thermo Fisher). Five to seven days after transfection, culture supernatants were harvested, filtered, and incubated with Protein G Sepharose (GE Life Sciences) overnight at 4°C. Antibodies were eluted from chromatography columns using 0.1 M glycine (pH = 3.0) and buffered in 1 M Tris (pH = 8.0). Subsequent buffer exchange to PBS and antibody concentration was performed using Amicon 30 kDa spin membranes (Millipore). Antibodies were filter-sterilized using Ultrafree-CL or Ultrafree-MC 0.22 µm membranes (Millipore) and stored at 4°C.

Serum samples from HIV-1-infected individuals were heat-inactivated at 56 °C for 40 min and incubated with Protein G Sepharose (GE Life Sciences) overnight at 4 °C. IgGs were eluted from chromatography columns using 0.1 M glycine (pH = 3.0) into 0.1 M Tris (pH = 8.0). Buffer was exchanged to PBS through Amicon 30 kDa spin membranes (Millipore). Concentrations of purified IgGs were determined by UV/Vis spectroscopy (A280) on a Nanodrop 2000 and samples were stored at 4 °C.

Blood samples were obtained under protocols approved by the Institutional Review Board (IRB) of the University of Cologne (protocols 13-364 and 16-054) and the local IRBs and all participants provided written informed consent. HIV-1 infected patients were recruited at private practices and/or hospitals in Germany. Plasma samples were obtained and stored at -80°C until further use. Prior to IgG isolation, plasma samples were heat-inactivated at 56°C for 40 minutes. IgGs were isolated through an overnight incubation with Protein G Sepharose (GE Life Sciences) at 4°C, followed by elution with 0.1 M glycine (pH=3.0) using chromatography columns. The eluted IgGs were buffered in 1 M Tris (pH=8.0) and then underwent buffer exchange to phosphate-buffered saline (PBS) and concentration using Amicon 30 kDa spin membranes (Millipore). The purified IgGs were stored at 4°C until further use.