Shirasu Y, Shimada Y, Takemura K, Izumi T, Miyazaki H
Pharmaceutical Laboratory, Kirin Brewery Co., Ltd., Gunma, Japan.
Hybridoma. 1988 Oct;7(5):485-93. doi: 10.1089/hyb.1988.7.485.
Three murine monoclonal antibodies (MAbs) against human pancreatic elastase 2 (ELS-2) were produced by hybridomas obtained from the fusion of murine myeloma cells, P3X63-Ag8.-653, with the splenocytes of mice immunized with recombinant human proELS-2 (r-proELS-2) and purified natural ELS-2. These MAbs were found to recognize the different epitopes of ELS-2 based on a competitive binding assay. In addition, one of the MAbs, E19, reacted with the activation peptide of ELS-2. Western blot analysis performed under nonreducing conditions indicated that all MAbs bound only to ELS-2 in pancreatic juice in addition to natural ELS-2. Reduced ELS-2 on a Western blot could not be detected with E19, indicating that the activation peptide remained attached to active ELS-2 via a disulfide bond even after tryptic activation. Another MAb, B4, inhibited the enzymatic activity of ELS-2, indicating that B4 recognized the active site or its vicinity to ELS-2.
通过将鼠骨髓瘤细胞P3X63 - Ag8.-653与用重组人原弹性蛋白酶2（r - proELS - 2）和纯化的天然弹性蛋白酶2（ELS - 2）免疫的小鼠脾细胞融合得到的杂交瘤，产生了三种抗人胰腺弹性蛋白酶2（ELS - 2）的鼠单克隆抗体（MAb）。基于竞争结合试验发现这些单克隆抗体识别ELS - 2的不同表位。此外，其中一种单克隆抗体E19与ELS - 2的激活肽发生反应。在非还原条件下进行的蛋白质印迹分析表明，除了天然ELS - 2外，所有单克隆抗体仅与胰液中的ELS - 2结合。用E19在蛋白质印迹上检测不到还原型的ELS - 2，这表明即使在胰蛋白酶激活后，激活肽仍通过二硫键与活性ELS - 2相连。另一种单克隆抗体B4抑制ELS - 2的酶活性，表明B4识别ELS - 2的活性位点或其附近区域。
