Figure 8.

The LDH assay was used to determine the anti-CD20/CD3 BsNb-mediated killing activity of PBMCs against tumor cells. Raji cells were cultured in a PerkinElmer Cell Carrier for 12 h. PBMCs were added to each group in three parallel wells at ratios of effector: target = 5:1 and effector: target = 10:1, and anti-CD20/CD3 BsNb was added to the experimental group to a final concentration of 40 μg/mL, 20 μg/mL, 10 μg/mL, 5 μg/mL, 2 μg/mL, and 1 μg/mL. Then, 100 μL RPMI 1640 medium was added to blank wells, 50 μL of Raji cells + 50 μL of PBMCs were added to sample control wells, and 50 μL of Raji cells + 50 μL of PBMCs + 10 μL of lysate were added to maximum enzyme activity control wells and incubated for 24 h at 37 °C. Then, 10 μL of lysate was added to the target cell maximum release wells 1 h before the end of incubation and mixed thoroughly. The optical density was measured at OD490 nm with a microplate reader, and the mortality rate of Raji cells was calculated using the following common formula: mortality rate (%) = {[(sample well OD490 nm-blank well OD490 nm) − (sample control well OD490 nm-blank well OD490 nm)]/(maximum enzyme activity control well OD490 nm-blank well OD490 nm)} × 100% (** p < 0.01).