LRRK2
外周血单个核细胞
激酶
分析物
抗体
磷酸化
药理学
医学
生物
疾病
化学
免疫学
体外
生物化学
内科学
帕金森病
色谱法
作者
Robert M. Umek,Amit S. Adhikari,Jill Dunty,Priya Ranganathan,Matthew Bess,Eugene Perelshteyn,Jacob N. Wohlstadter
标识
DOI:10.1016/j.jalz.2017.06.1280
摘要
Leucine-rich repeat kinase 2 (LRRK2) is the strongest known genetic contributor to Parkinson's disease (PD). LRRK2 protein is a kinase and the subject of numerous drug development programs throughout the pharmaceutical industry. As such, assays that quantify LRRK2 and LRRK2 specifically phosphorylated serine 935 (pS935 LRRK2) are desirable to screen for inhibitors and to quantify pharmacokinetic targets. The development of such assays has proven challenging since there is a paucity of antibodies available against these targets. Using MSD's U-PLEX® platform, we have developed sandwich immunoassays for both analytes and shown their suitability for use with human peripheral blood mononuclear cells (PBMCs). Antibody pairs were selected and their concentrations optimized to create the most robust assays possible from the available antibodies. As test samples, PBMCs were prepared from human donors and treated with or without a LRRK2 kinase inhibitor. Cell lysates were prepared from the PBMCs and analyzed with the new assays. Cells treated with the inhibitor showed a time-dependent decrease in the abundance of pS935 LRRK2. A modest decrease in “total” LRRK2 was also noted. The results confirm that the pS935 LRRK2 assay is specific for quantifying reversible phosphorylation of LRRK2. Immunoassays for LRRK2 and pS935 LRRK2 were successfully developed. The assays are capable of high throughput and are compatible with PBMCs, a sample type common to clinical research. The assays should prove useful for LRRK2 drug development programs both during pre-clinical research and to provide ancillary data in support of clinical studies. The reported research was funded by the Michael J. Fox Foundation for Parkinson's Research.
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