Protocol for using an ELISA to detect total α-synuclein levels in Drosophila melanogaster lines expressing human α-synuclein point mutations

Abstract In Parkinson’s disease (PD), aggregation of alpha-synuclein (α-syn) contributes to neuronal dysfunction and death, particularly in dopaminergic neurons, driving disease progression. Several pathogenic point mutations have been identified in human α-syn such as A30P, E46K, H50Q, G51D, A53T and A53E that have been associated with PD. In this study, a sandwich ELISA assay was developed to quantify total α-syn levels in various Drosophila melanogaster genotypes expressing A30P, E46K, G51D and A53T . Using this approach, we compared α-syn levels across wild-type (WT) and mutant forms of the protein. The E46K and A53T mutations exhibited higher total α-syn concentrations compared to WT and the G51D mutation. In addition to characterizing mutationdependent differences in α-syn levels, this assay was applied to evaluate smallmolecule modulators that inhibit α-syn aggregation. These findings demonstrate that the ELISA-based approach provides a useful platform for quantifying α-syn and assessing potential therapeutic compounds targeting αsyn pathology.