Development of monoclonal mouse antibodies that specifically recognize pancreatic polypeptide
Development of monoclonal mouse antibodies that specifically recognize pancreatic polypeptide
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DOI：
10.1507/endocrj.ej18-0441
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发表时间：
2019-01-01
影响因子：
2
通讯作者：
Fujitani, Yoshio
中科院分区：
文献类型：
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作者：
Hara, Akemi;Nakagawa, Yuko;Fujitani, Yoshio
胰多肽（PP）是由Ppy基因编码的36个氨基酸的肽，其由位于胰岛周围的小细胞群产生。由于神经肽Y家族成员之间的高度氨基酸序列相似性，目前可获得的针对PP的抗体对PP没有令人信服的特异性。在这里，我们报告的小鼠单克隆抗体，特异性结合PP的发展。我们产生了Ppy敲除（Ppy-KO）小鼠，其中Ppy编码区被Cre重组酶替换。用小鼠PP肽免疫Ppy-KO小鼠，并分离产生抗PP抗体的稳定杂交瘤细胞株。首先，在酶联免疫吸附测定中选择阳性克隆用于与偶联牛血清白蛋白的PP的反应性。在筛选期间，排除产生与肽YY（PYY）交叉反应的抗体的杂交瘤克隆。在第二次筛选中，选择杂交瘤克隆，其中它们的培养基在Ppy-KO胰岛中不产生信号，但在野生型胰岛的外周区域中检测到特异性细胞。进一步的研究表明，选择的单克隆抗体（23- 2D 3）特异性识别PP产生细胞，不仅在小鼠，而且在人类和大鼠胰岛。本研究开发的具有高结合特异性的PP单克隆抗体将为进一步研究PP的表达谱和生理作用奠定基础。
Pancreatic polypeptide (PP) is a 36-amino acid peptide encoded by the Ppy gene, which is produced by a small population of cells located in the periphery of the islets of Langerhans. Owing to the high amino acid sequence similarity among neuropeptide Y family members, antibodies against PP that are currently available are not convincingly specific to PP. Here we report the development of mouse monoclonal antibodies that specifically bind to PP. We generated Ppy knockout (Ppy-KO) mice in which the Ppy-coding region was replaced by Cre recombinase. The Ppy-KO mice were immunized with mouse PP peptide, and stable hybridoma cell lines producing anti-PP antibodies were isolated. Firstly, positive clones were selected in an enzyme-linked immunosorbent assay for reactivity with PP coupled to bovine serum albumin. During the screening, hybridoma clones producing antibodies that cross-react to the peptide YY (PYY) were excluded. In the second screening, hybridoma clones in which their culture media produce no signal in Ppy-KO islets but detect specific cells in the peripheral region of wild-type islets, were selected. Further studies demonstrated that the selected monoclonal antibody (23-2D3) specifically recognizes PP-producing cells, not only in mouse, but also in human and rat islets. The monoclonal antibodies with high binding specificity for PP developed in this study will be fundamental for future studies towards elucidating the expression profiles and the physiological roles of PP.