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Figure 1. Map of the pBudCE4.1 expression vector containing the mouse-human chimeric antibody (c-1T0). Light chain sequences were introduced in XhoI and KpnI sites after PEF-1α promoter and heavy chain sequences were introduced into SalI and BamHI sites after the PCMV promoter.
(Figure omitted. See article PDF.)
Figure 2. PCR amplification of VH-CH and VL-CL genes for construction of the chimeric antibody. (A & B) Mouse VH and VL chain genes, and (C & D) human constant region heavy chain of IgG1 (C g1 ) and klight chains (C k) were amplified from cDNA of the mouse hybridoma 1T0 clone and human peripheral blood mononuclear cells, respectively. (E) Vk-Ckand (F) VH-Cg1 fragments were amplified by splice overlap extension PCR as described in the materials and methods section. The PCR products were run on 1% agarose gel. (A, C, D & F) A 100-bp Plus DNA ladder (Sinaclon, Tehran, Iran) and (B & E) DNA molecular weight marker IX (Roche, Mannheim, Germany) were used.
(Figure omitted. See article PDF.)
Figure 3. Measurement of chimeric anti-HER2 monoclonal antibody in culture supernatant of transfected cells. (A) Titration of trastuzumab and (B) c-1T0 culture supernatant on the recombinant extracellular region of HER2 is shown. The supernatant of mock-transfected CHO cell line was used as a control (dotted line). OD: Optical density.
(Figure omitted. See article PDF.)
Figure 4. SDS-PAGE electrophoresis pattern of chimeric antibody. SPG-purified c-1T0 and mouse 1T0 IgG preparations were separated on 10% gel in nonreducing (Lanes 1 and 3) and reducing (Lanes 2 and 4) conditions. MW: Molecular weight ladder (Sinaclon, Tehran, Iran).
(Figure omitted. See article PDF.)
Figure 5. Western blot analysis of chimeric antibody. NR and R forms of recombinant HER2 extracellular protein were separated on 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blotted with c-1T0 (Lanes 3 and 6), mouse 1T0 (Lanes 2 and 5) and trastuzumab (Lanes 1 and 4) and then visualized by enhanced chemiluminescence, as described in the materials and methods section. NR: Nonreduced; R: Reduced.
(Figure omitted. See article PDF.)
Figure 6. Affinity constant determination of mouse 1T0 and chimeric c-1T0. Experimental dose-response curves for (A) mouse 1T0 and (B) chimeric c-1T0 monoclonal antibodies at three different concentrations of...