Figure 2

Flow cytometry and immunoprecipitation with ScFv 800E6 produced in E. coli and transgenic plants. Lysates (50 μl) from bacteria expressing either ScFv800E6 or ScFvαCTV, and the parental mAb 800E6 were incubated with 32D-ErbB-2 transfectants and ErbB-2-negative 32D cells (panels A and B, respectively), and revealed by an FITC-labeled antibody to whole murine Ig. Panel C: two-fold dilutions of bacterial lysates containing ScFv800E6 were tested as above on ErbB-2⁺ SK-BR-3 cells. ScFvαCTV and mAb 800E6 are reported for comparison. Panel D: metabolically radiolabeled SK-BR-3 cells were lysed and immunoprecipitated with the indicated antibodies and ScFvs. Asterisks mark a group of closely migrating background bands of unknown origin present in several lanes. These are unlikely to represent MHC class I heavy chains that are poorly, if at all, expressed by SK-BR-3 cells [30]. Panel E: Lysates (50 μl) from tobacco plantulas stably (stbl) or transiently (trns) expressing ScFv800E6, and from transiently mock-infected plants (as a representative negative control) were incubated with SK-BR-3 cells, and ScFv binding revealed by flow cytometry as above.