Gregor M, Riecken E O
Gastroenterology. 1985 Sep;89(3):571-80. doi: 10.1016/0016-5085(85)90453-6.
Hybridoma technology has been successfully applied to the production of monoclonal antibodies against a variety of small soluble peptides. We report herein for the first time on the development of monoclonal antibodies to pancreatic glucagon. Twenty-three stable positive hybridomas were detected by radioimmunoassay from five separate fusions and cloned by the limiting dilution method. Four selected monoclonal antibodies were all of the IgG 2a subclass type kappa and bound to protein A. One monoclonal antibody (23.8B6) was shown to be directed toward the C-terminal region and another (23.6B4) toward the N-terminal to central region of the glucagon molecule. These antibodies did not cross-react with any of the other peptides tested. Two further monoclonal antibodies (23.4A1, 22.3A6) reacted with the C-terminal third of the glucagon molecule and showed a cross-reaction with the structurally related gastric inhibitory polypeptide of 0.7% and 9.1%, respectively. All but the C-terminal monoclonal antibody 23.8B6 showed a marked cross-reaction with ileal extracts. The N-terminally directed monoclonal antibody 23.6B4 was of sufficient avidity for use in the radioimmunoassay of pancreatic glucagon and gut glucagon-like immunoreactivity in tissue extracts, being sensitive to changes of pancreatic glucagon of 2.0 fmol/tube at a final titer of culture supernatant of 1:1.4 X 10(5). In gel permeation chromatography of intestinal extracts, two major peaks were detectable (Kav 0.27 and 0.54). The present findings show that monoclonal antibodies provide sensitive tools for detecting pancreatic glucagon and gut glucagon-like immunoreactivity. They will be valuable immunoreactants for the development of immunoradiometric assays as well as for large-scale immunoaffinity purification of gut glucagon-like immunoreactivity.
杂交瘤技术已成功应用于生产针对多种小分子可溶性肽的单克隆抗体。我们在此首次报告抗胰高血糖素单克隆抗体的研制情况。通过放射免疫测定法从五次独立融合中检测到23个稳定的阳性杂交瘤，并采用有限稀释法进行克隆。所选的四种单克隆抗体均为IgG 2a亚类κ型，且能与蛋白A结合。其中一种单克隆抗体（23.8B6）被证明针对胰高血糖素分子的C末端区域，另一种（23.6B4）针对N末端至中央区域。这些抗体与所测试的任何其他肽均无交叉反应。另外两种单克隆抗体（23.4A1、22.3A6）与胰高血糖素分子的C末端三分之一反应，并分别与结构相关的胃抑制多肽有0.7%和9.1%的交叉反应。除C末端单克隆抗体23.8B6外，所有抗体均与回肠提取物有明显交叉反应。N末端导向的单克隆抗体23.6B4具有足够的亲和力，可用于组织提取物中胰高血糖素和肠胰高血糖素样免疫反应性的放射免疫测定，在培养上清液最终滴度为1:1.4×10⁵时，对2.0 fmol/管的胰高血糖素变化敏感。在肠道提取物的凝胶渗透色谱中，可检测到两个主要峰（洗脱体积0.27和0.54）。目前的研究结果表明，单克隆抗体为检测胰高血糖素和肠胰高血糖素样免疫反应性提供了灵敏工具。它们将是免疫放射测定法开发以及大规模免疫亲和纯化肠胰高血糖素样免疫反应性的有价值的免疫反应物。
