首页> 外文期刊>Analytical Biochemistry: An International Journal of Analytical and Preparative Methods >An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus
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An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus

机译:蛋白质芯片技术在牡蛎蘑菇球形病毒单克隆抗体筛选中的应用

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The oyster mushroom spherical virus (OMSV) is a causative agent of dieback disease in the oyster mushroom, Pleurotus ostreatus. Outbreaks of this virus occasionally result in serious disease that is associated with hefty economic losses. Thus, the detection and removal of OMSV-infected spawn is considered to be a crucial step for the stable production of P. ostreatus. For the detection of OMSV, we attempted to generate monoclonal antibodies (mAbs) against an RNA polymerase domain (RPD) of an OMSV protein. In an effort to simplify the laborious multistep mAb screening process, we developed a protein microarray on a slide glass that is chemically modified with the RPD protein. The culture supernatants of 87 hybridoma cells, which were prepared from the fusion of RPD-immunized mouse spleen cells with myeloma cells, were spotted onto the RPD-coated microarray. The binding of mAb to RPD was detected via Alexa 488 dye-labeled anti-mouse immunoglobulin G (IgG) as a secondary antibody. Of 87 samples, 13 evidenced a significant level of fluorescence signal intensity. Subsequent immunoblot analysis revealed that the specificity of each mAb against RPD coincided with the corresponding fluorescence signal intensity, thereby indicating the effectiveness of the protein microarray in mAb screening. (C) 2007 Elsevier Inc. All rights reserved.
机译:牡蛎蘑菇球形病毒(OMSV)是牡蛎蘑菇侧耳属中回死病的致病因子。这种病毒的爆发有时会导致严重的疾病,并带来巨大的经济损失。因此,检测和去除OMSV感染的产卵被认为是稳定生产平菇的关键步骤。对于OMSV的检测,我们尝试生成针对OMSV蛋白的RNA聚合酶结构域(RPD)的单克隆抗体(mAb)。为了简化繁琐的多步mAb筛选过程,我们在载玻片上开发了一种蛋白质微阵列,该载玻片经过RPD蛋白化学修饰。将由RPD免疫的小鼠脾细胞与骨髓瘤细胞融合制备的87个杂交瘤细胞的培养上清液点样到RPD包被的微阵列上。通过Alexa 488染料标记的抗小鼠免疫球蛋白G(IgG)作为二抗检测mAb与RPD的结合。在87个样本中,有13个样本显示出显着水平的荧光信号强度。随后的免疫印迹分析表明,每种单克隆抗体对RPD的特异性与相应的荧光信号强度一致,从而表明了蛋白质微阵列在单克隆抗体筛选中的有效性。 (C)2007 Elsevier Inc.保留所有权利。

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