Wen-Li et al. Journal of Experimental & Clinical Cancer Research 2012, 31:92 http://www.jeccr.com/content/31/1/92 Page 5 of 9 immunogen and the human ATPase β subunit, which had been expressed in prokaryotes. After several fusion experiments, hundreds of monoclonal hybridoma cells were obtained. One immunoglobulin G1 (IgG1) hybri- doma clone, named McAb7E10, recognized both the native and recombinant ATPase β subunit. In Western blot analysis, the McAb7E10 antibody identified a single band corresponding to the molecular mass of the ATPase β subunit, and did not cross react with the ATPase α subunit (Figure 2A). The affinity of McAb7E10 to the recombinant ATPase β subunit was evaluated using BIAcore, and the dissociation constant was KDMcAb7E10 = 3.26E–10 (Figure 2B), which is higher than the KD of 4.24E–9 of the previously characterized ATPase β subunit antibody McAb178-5 G10 [3]. McAb7E10 inhibits cell surface ATP generation in AML cells To examine the inhibitory effect of the antibody on ATP synthesis, a cell surface ATP generation assay was per- formed. Results showed that McAb7E10 antibody signifi- cantly inhibited ATP synthesis in AML cells. The relative inhibitory rates in 25, 50 and 100 ug/mL McAb7E10 treated MV4-11 cells were 14.1%, 23.1% and 25.0%, in HL-60 cells were 16.1%, 28.1% and 29.3% re- spectively (Figure 3A, 3B). The maximal inhibition of McAb7E10 to MV4-11 and HL-60 cells was ~30% (300 μg/mL), and the maximal inhibition of oligomycin to both cells was ~80% (300 μg/mL). McAb7E10 inhibits AML cell proliferation and induces apoptosis in AML cells This study provides evidence that McAb7E10 preferen- tially binds to the cell surface ATPase β subunit, and can inhibit cell proliferation and induce apoptosis in MV4-11 and HL-60 AML cells. The effect of McAb7E10 on the proliferation of MV4-11 and HL-60 cells was evaluated using the MTT assay. Compared to control mouse IgG treated cells, after 120 h, the relative inhibitory rates in 5, 10 and 50 ug/mL McAb7E10 treated MV4-11 cells were 24.5%, 44% and 69.6%, respectively (Figure 3C). After 120 h, the relative inhibitory rates in 5, 10 and 50 ug/mL McAb7E10 treated HL-60 cells were 39.4%, 62.1% and 81.9%, respectively (Figure 3D). These results indi- cate that McAb7E10 can significantly inhibit the pro- liferation of AML cells in vitro. Using cell cycle analysis and Annexin V staining, a subpopulation of cells before the G1 population was detected after treatment with McAb7E10, indicating cells with abnormal nuclei which can be considered to be apoptotic and dead cells. The relative rate of apop- tosis in 5, 10 and 50 ug/mL McAb7E10 treated MV4-11 cells was 3.6 ± 0.83%, 8.4 ± 1.69% and 17.3 ± 2.56% compared to 1.5% ± 0.85% in mouse IgG treated cells (p < 0.01, Figure 4A, 4B). The relative rate of apoptosis in 5, 10 and 50 ug/mL McAb7E10 treated HL-60 cells was 5.5 ± 2.37%, 11.3 ± 3.62% and 19.9 ± 3.31% compared to 1.56% ± 0.97% in mouse IgG treated cells (p < 0.01, Figure 4A, 4C). To determine whether McAb7E10 can Figure 2 Production and characterization of McAb7E10. A monoclonal antibody with a high valency against F1F0 ATPase β subunit was developed and named McAb7E10. (A) In Western blot analysis, the McAb7E10 antibody detected a single immunoreactive band in HUVEC protein lysate (lane 1) and recombinant ATPase β subunit protein (lane 2), but did not detect recombinant human ATPase α subunit protein (lane3). (B) The affinity of McAb7E10 to recombinant ATPase β subunit was evaluated using BIAcore. The affinity of McAb7E10 to the recombinant ATPase β subunit was evaluated using BIAcore, and the dissociation constant was KDMcAb7E10 = 3.26E–10.