Screening of cell-internalizing mAbs (ELISA and cytotoxicity assay). To screen for cell-internalizing mAbs, 315 clones per antigen were analyzed by ELISA and cytotoxicity assay. (A) Result for Robo4, (B) Result for VEGFR2. Monoclonal scfv-PSIFs were induced in TG1 supernatant. The binding properties and cytotoxicities to MS1 cells were then assessed by an ELISA and WST-8 assay, respectively. E, ELISA results; C, WST-8 assay results. Individual results from ELISA (OD = 0.8 or 0.5∼0.0) and WST-8 assay (cytotoxicity = 30%∼0%) were mapped in grayscale densities. The 24 candidates against Robo4 and 17 candidates against VEGFR2 are indicated by the underline (ELISA OD ≥0.2 and cytotoxicity ≥20%). After omitting redundant clones by sequencing, 1 cell-internalizing mAb and 2 low-internalizing mAbs against mRobo4, and 2 cell-internalizing mAbs and 14 low-internalizing mAbs against mVEGFR2 were identified.