Figure 2.

GrB/scFvMEL specifically binds to the gp240 antigen-positive melanoma cell lines, as assessed by ELISA. Ninety-six-well ELISA plates containing adherent melanoma cells (5 x 10⁴ cells/well) were blocked by addition of a solution containing 5% BSA for 1 hour. To detect the binding activity of GrB/scFvMEL, cells were incubated with purified GrB/scFvMEL at various concentrations for 1 hour at RT. After washing, the cells were incubated with rabbit anti-scFvMEL antibody, followed by the addition of HRP-GAR antibody. Finally, the substrate solution ABTS containing 1 µl/ml 30% H₂O₂ was added to the wells. Absorbance at 405 nm was measured after 30 minutes. GrB/scFvMEL bound to high-level gp240 antigen-expressing melanoma A375-M, MEL-526, and TXM-18 cells, but not to TXM-1 cells, which has a very low-level expression of the gp240 antigen.