Fusion and HAT selection of HA-AP-EGF-R-containing Sp2/0 cells with spleen lymphocytes from ovalbumin immunized Balb/c mice

The hybridoma technique was used to analyze the cell surface expression behavior of the HA-AP-EGF-R fusion protein after fusion of a transfected Sp2/0 cell with spleen lymphocytes from an ovalbumin immunized Balb/c mouse. Immunization was performed according to the relevant national and international guidelines. The study was approved by the Ministry of Environment, Health and Consumer Production of the Federal State of Brandenburg (reference number V3-2347-A16-4-2012). Briefly, Balb/c mice (8–12 wks old) were immunized intraperitoneally with 100 µg ovalbumin (Sigma-Aldrich, USA) in 200 µL PBS supplemented with 50 µL Complete Freund´s Adjuvant (CFA). The fusion was conducted according to Köhler and Milstein¹¹ with an electrofusion modification⁴⁰. Briefly, spleen lymphocytes were mixed in a ratio of 3 to 1 with transfected myeloma cells that bear the artificial cell surface fusion protein and were washed three times with fusion buffer (125 mM NaCl, 5 mM KCl, 4 mM CaCl₂, 2.5 mM MgCl₂, 5 mM Tris-HCl pH 7.4, 0.2 µm). The cell pellet was dissolved in 200 µL fusion buffer and filled up to a final volume of 400 µL with 25% (w/v) PEG8000 in fusion buffer. The cell suspension was transferred into a fusion cuvette and placed into the electro fusion device. The cells were pulsed with 600 V for 25 ms. Three minutes post pulse the cell suspension was transferred into 37 °C pre-warmed full growth media supplemented with 20% (v/v) FCS and cultured for 5 h at 37 °C and 95% humidity under 6% CO₂. Following that, HAT medium (hypoxanthine; 27.24 µg/mL, azaserine; 2 µg/mL and thymidine; 7.76 µg/mL) was added to the cell suspension. Finally the cell suspension was transferred into T75 flasks and further cultured at 37 °C and 95% humidity under 6% CO₂. HAT selection was performed for 10 days. Growing hybrids were then checked for HA-AP-EGF-R surface receptor expression and secretion of ovalbumin-specific antibodies as described below.