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. 2017 Oct 7;8(55):94210–94222. doi: 10.18632/oncotarget.21618

Figure 1. Screening and identification of positive fusion cell clones and generation of chimeric monoclonal antibody ROR1-cFab.

Figure 1

(A) ELISA. ELISA was performed to screen 31 candidate fusion cell clones after three rounds of sub-clone screening. NC, a negative control. (B) Agarose gels of PCR amplification of the light chain. Lane 1, VL; Lane 2, CL; Lane 3, VL combined with CL; M, a DNA maker. (C) Agarose gels of PCR amplification of the heavy chain. Lane 1, VH; Lane 2, CH1; Lane 3, VH combined with CH1; M, a DNA maker. (D) Agarose gels of PCR amplification of pETDuet-ROR1-cFab. Lane 1, the plasmid of pETDuet without restriction endonuclease digestion; Lane 2, the plasmid of pETDuet-L; Lane 3, NcoI/HindIII were used for double digesting the pETDuet-L plasmid; Lane 4, the plasmid of pETDuet-L-H (pETDuet-ROR1-cFab); Lane 5, NdeI/kpnI were used for double digestion of the pETDuet-ROR1-cFab plasmid; M, a DNA marker. (E) Agarose gels of PCR amplification of the light and heavy chain of pETDuet-ROR1-cFab. Lane 1, L; Lane 2, Fd; M, a DNA maker. (F) Coomassie blue staining of a SDS-PAGE gel and (G) Western blot. Detection of expression of the Fab fragment in E. coli. Lane 1, supernatant of sonicated lysate of untransfected E. coli BL21, a negative control; Lane 2, sediment of sonicated lysate of untransfected E. coli BL21, a negative control; Lane 3, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli; Lane 4, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli; Lane 5, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; Lane 6, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; M, a protein marker. (H) Coomassie blue staining and (I) Western blot. Detection of the purification efficiency of the Fab fragments. Lane 1, the Fab fragments was purified by the Protein L affinity column; lane 2, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; lane 3, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; lane 4, the flow through the Protein L affinity column; M, a protein marker.