Naoto Nakamura, Mariko Shimokawa, Kazuyoshi Miyamoto, Shintaro Hojyo, Hiroyuki Horiuchi, Shuichi Furusawa and Haruo Matsuda
Journal of Immunological Methods, Volume 280, Issues 1-2 , September 2003, Pages 157-164
Laboratory of Immunobiology, Department of Molecular and Applied Biosciences, Graduate School of Biosphere Sciences, Hiroshima University, 1-4-4 Kagamiyama, Higashi, Hiroshima 739-8528, Japan
Received 28 August 2002; revised 15 January 2003; accepted 11 April 2003. ; Available online 6 September 2003.
We previously reported the development of chicken monoclonal antibodies (mAb) against mammalian-conserved molecules by cell fusion and phage display using the mouse mAb expression vector pPDS. However, chicken hybridomas produce relatively small amounts of antibody when compared with mouse hybridomas, and application of the pPDS may be limited in two-antibody assays with a mouse mAb because it contains mouse C as a detection tag. To circumvent the above problems, two expression vectors were established and used to produce a functional recombinant chicken mAb. These vectors, which were designed to accommodate a single chain fragment of the variable region (scFv) of the antibody, contained a chicken C and FLAG with or without 6¥ histidine sequences in the 3¢ terminus of the scFv to serve as detection and purification tags. In this study, a prion protein (PrP)-specific chicken mAb (HUC2-13) was expressed as phage-displayed and soluble scFv mAb forms by using these vectors. The scFv mAbs expressed by these vectors exhibited the same antigen-binding specificity to PrP as that of the original HUC2-13, could be purified with ease, and used in combination with a mouse mAb. These results indicate that the methods described herein offer an alternative to chicken mAb production from hybridomas and immunized chicken splenocytes, and may contribute to the use of chicken mAb reagents in numerous fields.
Author Keywords: Chicken monoclonal antibodies; Cell fusion; Phage display; Expression vectors
Abbreviations: scFv, single chain fragment of the variable region; mAb, monoclonal antibody; VH, variable region of the heavy chain; V, variable region of the light chain; PrP, prion protein; CHB, reverse primer for heavy chain variable region; CHSF, forward primer for heavy chain; CLSB, reverse primer for chain; CLF, forward primer for chain; CRB, reverse primer for reamplification of scFv fragment
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