Figure 6

Transfer of the VEGF MAR-HAC vector to CHO K1 cells promotes antibody production. (A) Two-color FISH analysis of the VEGF MAR-HAC vector in G418-resistant CHO K1 clones was performed with digoxigenin-labeled hCot-1 DNA (red) and biotin-labeled anti-VEGF antibody gene (green). The arrow indicates the HAC, and the inset shows enlarged images of the HAC (anti-VEGF antibody probe-specific green signals are indicated by arrows). The arrowheads indicate the three large metacentric chromosomes that identify CHO K1 cells. The left panel shows CHO K1R VEGF MAR-HAC cl.1. The right panel shows CHO K1R VEGF MAR-HAC cl.2. (B) Analysis of anti-VEGF antibody production with ELISA from CHO K1R VEGF MAR-HAC clones and CHO DG44 VEGF MAR-HAC clones from day 2 to day 8. Bars correspond to the means ± SD of three independent experiments. (C) Culture supernatants were analyzed by SDS-PAGE. The protein bands were stained by silver staining. MW, molecular weight markers; line 1, antibody purified from CHO DG44 VEGF MAR-HAC cl.2; line 2, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 3, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 4, commercial human anti-VEGF antibody (bevacizumab; 0.1 µg). Hc means heavy chain. Lc means light chain. (D) Western blotting of the 19.1 kDa VEGF 165 protein with the purified antibody from the CHO cell clones. Line 1, antibody purified from CHO DG44 VEGF MAR-HAC cl.2; line 2, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 3, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 4, commercial human anti-VEGF antibody.