All animal care and experimental protocols were performed in accordance with the guidelines for the care and use of laboratory animals at Chugai Pharmaceutical Co., Ltd. The protocol was approved by the Institutional Animal Care and Use Committee at Chugai Pharmaceutical Co., Ltd. Five New Zealand White rabbits (Kitayama Labes) were immunized four times with purified FLAG-tagged mouse PlxnA1 ectodomain. Then, the rabbits were humanely sacrificed by exsanguination under anesthesia, and their peripheral blood mononuclear cells and splenocytes were isolated. B cells positive for the production of anti-PlxnA1 were cultivated using the method reported by Seeber et al. [19 (link)] and were subjected to sequencing of the genes encoding the antibody variable regions. The DNA segment encoding each rabbit’s Ig variable region was inserted into an expression vector containing the constant region of various IgG species, including rabbit IgG/kappa, mouse IgG1/kappa, mouse IgG2a/kappa, or human IgG4/kappa, to express as recombinant IgG in FreeStyle293 cells. Recombinant IgGs were purified from the culture supernatants using rProtein A-Sepharose (GE Healthcare). Fab fragments were prepared and purified as previously described [20 (link)].
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