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[P3–070]: NOVEL ASSAYS FOR LRRK2 AND PS935 LRRK2
Author(s) -
Umek Robert M.,
Adhikari Amit,
Dunty Jill,
Ranganathan Priya,
Bess Matthew,
Perelshteyn Eugene,
Wohlstadter Jacob N.
Publication year - 2017
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2017.06.1280
Subject(s) - lrrk2 , peripheral blood mononuclear cell , kinase , analyte , antibody , phosphorylation , pharmacology , medicine , biology , disease , chemistry , immunology , in vitro , biochemistry , parkinson's disease , chromatography
Background:Parkinson’s disease (PD), like other neurological disorders, has been recognized to have a genetic component. Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene are recognized as genetic risk factors for familial PD, and may also represent causal factors in the more common sporadic form of the disease. The central role of LRRK2 in genetic and idiopathic PD has led to significant interest in further characterizing LRRK2 at both the genetic and protein levels. Methods: We developed an ultrasensitive digital immunoassay utilizing single molecule array (Simoa) technology for the purpose of measuring LRRK2 in human cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMC) as well as rat and human brain lysate. Limit of detection (LOD) was determined from recombinant protein calibration curves. Sensitivity and dilutional linearity was performed using human CSF and brain lysate to evaluate assay specificity, and spike recovery of recombinant LRRK2 protein was evaluated for measurement accuracy in sample matrix. Cross reactivity was also tested between LRRK2 and LRRK1 recombinant proteins. Using this digital ELISA, LRRK2 levels in PBMC and brain lysates were determined and compared with the results from Singulex Erenna platform. Results:The Simoa assay measured recombinant LRRK2 with a LOD of 0.2pg/mL. Average dilutional linearity was 111% for normal human CSF spiked with LRRK2 recombinant protein. Endogenous LRRK2 parallelism averaged 113% for rat brain lysates. Analyte spike recovery for human CSF ranged from 80% to 116% with a mean of 96%. No cross reactivity was observed between LRRK2 and LRRK1 recombinant proteins. The measured LRRK2 levels correlated between this digital ELISA and Erenna assay for both PBMCs and rat brain lysates. Conclusions:The ultrasensitive digital Simoa assay has potential to accurately quantify LRRK2 in PBMC and brain lysates. Data needs to be replicated with a larger data set including samples from patients diagnosed PD.