Total RNA was extracted from the hybridomas and reverse transcribed. The target sequences were amplified by PCR using primers targeted to the variable region of the heavy chain and the light chain of the human antibody. The sequences of the heavy and light chains were cloned into the pEHX1.1 vector and pELX2.2 vector, respectively [19 (link),23 (link),24 (link)]. A plasmid encoding both the heavy and light chains was constructed and transfected into Expi293 cells by using Expi Fectamine 293 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
At four days post-transfection, the human antibodies in the culture media were purified by using a Hi Trap rProtein A FF column (GE Healthcare, Chicago, IL, USA), and the automated chromatography system AKTA pure 25 (GE Healthcare, Chicago, IL, USA). The concentration of the purified antibodies was measured by using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
At four days post-transfection, the human antibodies in the culture media were purified by using a Hi Trap rProtein A FF column (GE Healthcare, Chicago, IL, USA), and the automated chromatography system AKTA pure 25 (GE Healthcare, Chicago, IL, USA). The concentration of the purified antibodies was measured by using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
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