Granulin specific monoclonal antibodies to investigate their expression and role

描述（由申请人提供）：美国有超过700万例痴呆症（约占人口的3%），其中约5%为额颞叶痴呆（FTD）。最近有研究表明，美国所有FTD中有5-10%是由前颗粒蛋白（PGRN）基因突变引起的。这伴随着患者生物体液中PGRN水平降低50%。PGRN是一种593氨基酸的生长和生存因子。PGRN的克隆和测序表明，它含有一个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半胱氨酸颗粒蛋白（grn）基序。6 Kda Grns是由PGRN加工产生的，PGRN是由弹性蛋白酶刺激的蛋白水解裂解产生的，并被分泌性白细胞蛋白酶抑制剂SLPI阻断。最近的证据表明，PGRN和grn都可能直接影响神经退行性疾病的过程，如阿尔茨海默病、帕金森病和某些类型的FTD。研究表明，PGRN和grn水平的平衡对于炎症导致的神经元保护/退化很重要。因此，PGRN和grn水平的测量可能揭示了大脑中正常与疾病相关过程的平衡。目前，与PGRN相比，对大脑中特定grn的功能和表达以及PGRN和grn与这些疾病之间可能的联系机制知之甚少。因此，开发特异性验证的检测系统来推进PGRN/grn领域在神经退行性疾病中的发展至关重要。首席研究员是公认的PGRN研究专家，特别是在癌症方面，并开发了临床验证的高质量单克隆抗体和检测方法，以测量患者组织和生物体液中的PGRN。她的PGRN检测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。然而，目前还没有可靠、敏感和特异性的方法来测量grn分子，从而阻碍了对grn在神经退行性疾病中的作用的理解。首席研究员正在申请RO3申请，以开发针对grn的单克隆抗体组合。该方法将针对7种不同grns的新切割位点，开发适合7种grns的夹心ELISA检测的单克隆抗体。具体而言，建议：1)开发适合生物样品夹心ELISA检测的针对7grn分子的高亲和力单克隆抗体；2)开发标准化实验室检测方法，在浓度小于100 pg/ml且不受PGRN干扰的情况下，特异性检测生物体液中的7grn。这些研究将是重要的，因为开发出有效的7种grns夹心ELISA试剂盒，它将使研究人员能够调查各种grns的存在和比例

DESCRIPTION (provided by applicant): 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 principal investigator 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 principal investigator 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.