Granulin specific monoclonal antibodies to investigate their expression and role

摘要 在美国有超过700万例痴呆症（约占人口的3%），其中约5%是额颞叶痴呆症。 痴呆（FTD）。最近已经证明，美国所有FTD的5-10%是由以下突变引起的： 前颗粒蛋白（PGRN）基因。这伴随着患者生物学检查中PGRN水平降低50%。 流体. PGRN是一种由593个氨基酸组成的生长和存活因子。PGRN基因的克隆和序列分析表明， 含有一个17个氨基酸的分泌信号肽和7个半6 kDa的grn重复序列，具有独特的 串联重复双富含半胱氨酸颗粒蛋白（GRN）基序。6 Kda Grns是通过处理 PGRN通过弹性蛋白酶刺激的蛋白水解切割和分泌性白细胞蛋白酶抑制剂阻断 SLPI。最近的证据表明，PGRN和grns都可能直接影响神经退行性疾病 过程，如阿尔茨海默氏病，帕金森氏病和某些类型的FTD。有人建议 PGRN和grn水平的平衡对于神经元保护/降解是重要的， 炎症因此，PGRN和grn水平的测量可以揭示正常的平衡， 与大脑中疾病相关的过程。目前，对该功能知之甚少， 与PGRN相比，脑中特异性grns的表达以及与PGRN相关的可能机制 PGRN和Grn对这些疾病的影响。因此，关键是开发特定的经验证的测定系统，以促进 PGRN/grn在神经退行性疾病中的研究进展PI是公认的PGRN研究专家 特别是在癌症中，并且已经开发出临床验证的高质量单克隆抗体和测定， 测量患者组织和生物体液中的PGRN。她的PGRN测定和试剂也用于 与两个神经退行性疾病卓越中心的合作研究。但没有 可靠、灵敏和特异的测定来测量GRN分子，从而妨碍了对GRN作用的理解。 在神经退行性疾病中的作用。PI请求在此RO 3应用程序中提供支持，以开发 GRN特异性单克隆抗体组合。该方法将靶向蛋白质的新切割位点。 七种不同的grn，以开发适用于7种grn中每一种的夹心ELISA测定的mAb。具体 目的：1）研制适用于夹心ELISA的特异性单克隆抗体 生物样品中的检测：2）开发标准化的实验室检测方法，用于特异性检测7种Grns 在生物流体中以低于100 pg/ml的浓度且不受PGRN干扰。这些研究 将是重要的，因为开发了经验证的夹心ELISA试剂盒，用于7种grns中的每一种， 研究人员调查CSF和其他生物制品中各种grns和PGRN的存在和比例， 神经退行性疾病患者的样本，如FTD。提供可以检测和测量的工具 并阐明这些重要的生物产品的作用也可能有助于揭示潜在的， 将它们用作治疗靶点或疾病或治疗监测的替代标记物。

Abstract Over 7 million cases of dementia exist in the US (~3% of the population) and of these ~5% are frontotemporal dementia (FTD). It has been demonstrated recently that 5-10% of all FTD in the US are caused by mutations in the Progranulin (PGRN) gene. This is accompanied with a 50% reduction of PGRN level in patients' biological fluids. PGRN is a 593 amino-acid growth and survival factor. Cloning and sequencing of PGRN showed that it contains a 17 amino-acid signal peptide for secretion and seven and a half 6 kDa grn repeats with a unique tandem repeat double cysteinyl rich granulin (grn) motif. The 6 Kda Grns are generated by processing of PGRN by a proteolytic cleavage stimulated by elastase and blocked by secretory leucocyte protease inhibitor SLPI. Recent evidence suggests that both PGRN and grns may directly influence neurodegenerative diseases process, such as Alzheimer's disease, Parkinson's disease and certain types of FTD. It has been suggested that the balance of PGRN and grn levels is important for neuron protection/degradation as a result of inflammation. Accordingly, the measurement of PGRN and grn levels may shed light on the balance of normal versus disease related processes in the brain. Currently, relatively little is known about the function and expression of specific grns in the brain when compared to PGRN and about the possible mechanisms linking PGRN and grn to these diseases. Thus, it is critical to develop specific validated assay systems to advance the PGRN/grn field forward in the neurodegenerative disease. The PI is a recognized expert in PGRN research particularly in cancer and has developed clinically validated high quality monoclonal antibodies and assays to measure PGRN in tissue and biological fluids in patients. Her PGRN assays and reagents are also used in collaborative studies with two centers of excellence in neurodegenerative disease. However, there are no reliable, sensitive and specific assays to measure grn molecules, thereby hampering understanding of the role of grns in neurodegenerative diseases. The PI is requesting support in this RO3 application to develop a portfolio of monoclonal antibodies specific to grn. The approach will be to target the neo-cleavage sites of the seven different grns to develop mAbs suitable for sandwich ELISA assay for each of the 7 grns. Specifically, it is proposed to: 1) Develop high affinity mAbs specific to each of 7 grn molecule suitable for sandwich ELISA detection in biological samples: 2) Develop standardized laboratory assays for specifically detecting the 7 grns in biological fluids at concentrations of less than 100 pg/ml and without interference by PGRN. These studies will be important as the development of validated sandwich ELISA kits for each of the 7 grns, it will enable researchers to investigate the presence and the ratio of various grns and PGRN in CSF and other biological samples from patients with neurodegenerative diseases such as FTD. Providing tools that can detect measure and elucidate the role of these important biological products may also be useful in revealing the potential to use them as therapeutic targets or surrogate markers for disease or therapy monitoring.