Intracellular light and heavy chains polypeptides of ethanol fixed cells were measured using a FITC-labeled goat anti-human kappa light chain antibody and a FITC-labeled goat anti-human IgG ( chain specific) antibody, respectively. early stages of cell line development. Additionally, we propose an approach using 25?cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones. Rev response element, central polypurine tract, human cytomegalovirus promoter, variable region of LC, constant region of LC (kappa), variable region of HC, constant region of HC, internal?ribosome entry site, neomycin phosphotransferase gene, woodchuck hepatitis virus posttranscriptional regulatory element. U3/3LTR: HIV-1 truncated 3 long terminal repeat Monoclonal antibodies Trastuzumab (trade name Herceptin), a humanized mAb specific for the human HER2 molecule, was purchased from Roche (Argentine). A biosimilar candidate to trastuzumab, named 5G4 and obtained from murine NS0 myeloma cells, was provided by Development Department of CIM (Havana, Cuba). Quantification of human IgG-expression levels by ELISA The human IgG-expression levels in 17-AAG (KOS953) cell culture supernatant were determined by sandwich ELISA. 96 well plates (High Binding, Costar, USA) were coated with 3?g/mL of a goat anti-human IgG ( chain specific) antibody (Sigma-Aldrich, USA) using coating buffer (Na2CO3/NaHCO3 0.1?M, pH 9.6). After a step of incubation at 4?C during 16?h, the plates were washed three times with washing buffer (phosphate buffered saline (PBS); Tween 20 at 0.05%, pH 7.5). The samples, diluted in blocking buffer (washing buffer and bovine serum albumin (BSA) at 0.25%), were applied to the plates and incubated at 37?C during 1?h. Then, the plates were washed three times with washing buffer and an alkaline phosphatase (AP)-conjugated goat anti-human IgG ( chain specific) antibody (Sigma-Aldrich, USA) was added. After another step of incubation at 37?C during 1?h, the plates were washed again and substrate was added (5?mg of p-nitrophenyl phosphate diluted in 5?mL of diethanolamine, pH 9.8). 30?min later, the reaction was stopped with NaOH 3?M and absorption was measured Rabbit polyclonal to AP4E1 at 405?nm on 17-AAG (KOS953) a microplate reader (Dialab, Austria). To quantify the expression levels, commercial trastuzumab was used as a standard (standard curve ranges from 3.9 to 125?ng/mL). Samples were analyzed in triplicate. In addition, another type of sandwich ELISA was used, allowing detection and quantification of antibody whole molecule. In this case, the samples were diluted in a different 17-AAG (KOS953) blocking buffer (washing buffer and 5% FBS) and it was used a horse-radish peroxidase (HRP)-conjugated goat anti-human kappa light chain antibody (Sigma-Aldrich, USA). The substrate was 5?mg of o-phenylenediamine dihydrochloride (OPD) in 10?mL of citrateCphosphate buffer (pH 4.2) and 20 L of H2O2 at 30%. Absorption was measured at 490?nm on a microplate reader (Dialab, Austria). Samples were analyzed in triplicate. Production and quantification of LVs LVs were produced by transfection of HEK-293T using lineal PEI (Sigma-Aldrich, USA) as previously described (Toledo et al. 2009) with some modifications. HEK-293T cells were cultured in a 75?cm2 T-flask in DMEM/F12-FBS medium until cells reached up to 70C80% confluence. The cells were co-transfected with one of the lentiviral transfer plasmids (pLW-CMV-trastuzumab?LC or pLV-CMV-trastuzumab?HC-IRES-Neo) and helper plasmids: pLP1, pLP2 and pLP VSV-G at a ratio of (2:1:1:1) (w:w:w:w) for 30?g of total DNA. Prior to transfection, cell culture supernatant was removed, the cells were washed with DMEM/F12 medium and 10?mL of this medium was added. In parallel, a mix of DNA, PEI and DMEM/F12 medium was prepared and added directly to the cells. After 6?h of incubation at 37?C in the presence of 5% CO2, 1?mL of FBS was added to the culture and the supernatant was harvested at 72?h post-transfection. The.