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

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 <i>A30P, E46K, H50Q, G51D, A53T</i> and <i>A53E</i> that have been associated with PD. In this study, a sandwich ELISA assay was developed to quantify total α-syn levels in various <i>Drosophila melanogaster</i> genotypes expressing <i>A30P, E46K, G51D and A53T</i> . Using this approach, we compared α-syn levels across wild-type (WT) and mutant forms of the protein. The <i>E46K</i> and <i>A53T</i> mutations exhibited higher total α-syn concentrations compared to WT and the <i>G51D</i> mutation. In addition to characterizing mutation-dependent differences in α-syn levels, this assay was applied to evaluate small-molecule 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.