Figure 5
Characterization of the artificial receptor functions on myeloma cells. (A) The biotin acceptor peptide (AP) is accessible on the surface of stably transfected myeloma cells. Transfected and non-transfected myeloma cells (1 × 106) were used for BirA mediated biotinylation. The cells in approaches III & IV were incubated with a mixture consisting of 5 mM MgCl2, 1 mM ATP, 10 µM Biotin and 0.789 µg BirA. The cells in approaches I, II, V, VI were incubated without BirA. To investigate whether BirA mediated biotinylation reaction was successful and specific, the cells (III, IV, V, VI) were treated with PE-conjugated streptavidin (2 µg/mL). After staining and washing cells were analyzed and 2.5 × 104 events were recorded by flow cytometry. Non-stained cell served as negative control. Dead cells were excluded by 7-AAD-staining. Analysis was performed by using BD CellQuest Pro software version 6.0. (B) The artificial receptor as bridge between phenotype and genotype. To investigate whether the HA-AP-EGF-R is a suitable tool to retain a soluble antibody at the surface of biotinylated transfected cells, a stable bridge was constructed between the receptor, an antigen and the antibody. Fluorescein was used as antigen which was bound to the surface as FITC-labeled SAV (2 µg/mL). The monoclonal mouse anti-fluorescein antibody B13-DE1 was used as antibody to be bound. As an isotype control a mouse anti-GST antibody (1 µg/1 × 106 cells) was used. A PE-conjugated F(ab)2 fragment of a donkey anti-mouse IgG diluted in PBS containing 0.5% BSA and 2 mM EDTA was used as indicator antibody. Dead cells were excluded by staining with 7-AAD and 1 × 104 events were recorded. Analysis was performed by using BD CellQuest Pro software version 6.0. (C) Antigen loaded HA-AP-EGF-R+ myeloma cells can be identified within a heterogeneous cell population. Transfected and non-transfected myeloma cells (1 × 106) were mixed together in a cell ratio of 1/1 followed by in vitro biotinylation reaction. The cells from approach III were incubated with a mixture of 5 mM MgCl2, 1 mM ATP, 10 µM Biotin and 0.789 µg BirA. The cells from approaches I and II were incubated without BirA. Following that the heterogeneous cell pool was incubated with a FITC-labeled SAV-conjugate (2 µg/mL). After this, the cells were labeled with B13-DE1 (1 µg/1 × 106 cells) and a PE-conjugated F(ab)2 fragment of a donkey anti-mouse IgG. Non-stained cells served as negative control and dead cells were excluded by staining with 7-AAD. 1 × 104 events were recorded and analysed by using BD CellQuest Pro software version 6.0.
