Nakamura Naoto, Shimokawa Mariko, Miyamoto Kazuyoshi, Hojyo Shintaro, Horiuchi Hiroyuki, Furusawa Shuichi, Matsuda Haruo
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.
J Immunol Methods. 2003 Sep;280(1-2):157-64. doi: 10.1016/s0022-1759(03)00204-7.
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 Ckappa 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 Clambda and FLAG with or without 6 x 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.
我们之前报道过，通过使用小鼠单克隆抗体（mAb）表达载体pPDS进行细胞融合和噬菌体展示，开发出了针对哺乳动物保守分子的鸡单克隆抗体（mAb）。然而，与小鼠杂交瘤相比，鸡杂交瘤产生的抗体量相对较少，并且在与小鼠mAb进行的双抗体检测中，pPDS的应用可能会受到限制，因为它含有小鼠Ckappa作为检测标签。为了规避上述问题，我们建立了两种表达载体，并用于生产功能性重组鸡mAb。这些载体被设计用于容纳抗体可变区的单链片段（scFv），在scFv的3'末端含有鸡Clambda和FLAG，FLAG带有或不带有6x组氨酸序列，用作检测和纯化标签。在本研究中，通过使用这些载体，将朊病毒蛋白（PrP）特异性鸡mAb（HUC2-13）表达为噬菌体展示的和可溶性scFv mAb形式。这些载体表达的scFv mAb对PrP表现出与原始HUC2-13相同的抗原结合特异性，易于纯化，并可与小鼠mAb联合使用。这些结果表明，本文所述方法为从杂交瘤和免疫鸡脾细胞生产鸡mAb提供了一种替代方法，并可能有助于鸡mAb试剂在众多领域的应用。
