Objectives: Lafora disease is a rare yet invariably fatal form of progressive neurodegenerative epilepsy resulting from mutations in the phosphatase laforin. Several therapeutic options for Lafora disease patients are currently being explored, and these therapies would benefit from a biochemical means of assessing functional laforin activity following treatment. To date, only clinical outcomes such as decreases in seizure frequency and severity have been used to indicate success of epilepsy treatment. However, these qualitative measures exhibit variability and must be assessed over long periods of time. In this work, we detail a simple and sensitive bioassay that can be used for the detection of functional endogenous laforin from human and mouse tissue.
Design and methods: We generated antibodies capable of detecting and immunoprecipitating endogenous laforin. Following laforin immunoprecipitation, laforin activity was assessed via phosphatase assays using para-nitrophenylphosphate (pNPP) and a malachite green-based assay specific for glucan phosphatase activity.
Results: We found that antibody binding to laforin does not impede laforin activity. Furthermore, the malachite green-based glucan phosphatase assay used in conjunction with a rabbit polyclonal laforin antibody was capable of detecting endogenous laforin activity from human and mouse tissues. Importantly, this assay discriminated between laforin activity and other phosphatases.
Conclusions: The bioassay that we have developed utilizing laforin antibodies and an assay specific for glucan phosphatase activity could prove valuable in the rapid detection of functional laforin in patients to which novel Lafora disease therapies have been administered.
Keywords: BSA; Bioassay; CBM; CSF; DSP; DTT; Epilepsy; Glucan phosphatase; HEK; HRP; LB; LD; Lafora body; Lafora disease; Laforin; Malachite green; PAS; TBS; Tris-buffered saline; bovine serum albumin; carbohydrate binding module; cerebrospinal fluid; dithiothreitol; dual specificity phosphatase; horseradish peroxidase; human embryonic kidney; mRIPA; modified RIPA buffer; pNPP; para-nitrophenylphosphate; pcDNA3.1 FLAG; pcDNA3.1 with N-terminal FLAG tag; periodic acid Schiff.
© 2013.