Generation and validation of polyclonal IgY antibodies for detection of endogenous Drosophila melanogaster insulin-like peptide 2

Insulin regulates essential metabolic processes, and disruptions in its signaling drive diverse metabolic, cardiovascular, neurodegenerative, and reproductive pathologies. Owing to the evolutionary conservation of insulin pathways, Drosophila melanogaster serves as a powerful model for studying insulin‑dependent glucose regulation. Among Drosophila insulin‑like peptides, DILP2 is highly similar in conserved sequence and structure to human insulin, which is a key regulator of carbohydrate storage and glucose homeostasis, making it a central focus of functional studies. Although anti-DILP2 antibodies have been employed in various studies, the limited availability of specific antibodies has constrained detailed functional and histological studies. In this study, we report the successful cloning, expression, and immunological evaluation of recombinant DILP2 to generate a highly specific IgY antibody. The dilp2 gene was cloned into the pET-21a vector and expressed in Escherichia coli , where the recombinant protein accumulated predominantly as inclusion bodies. Immunization with inclusion body-derived and refolded DILP2 resulted in different antibody responses. Although a higher total IgY level was observed in the single hen immunized with inclusion body-derived DILP2, detectable antigen-specific antibodies were obtained only following immunization with the refolded protein. The resulting IgY-DILP2 antibody specifically labeled insulin-producing cells (IPCs) in the adult fly brain. Further validation using a previously characterized dUCH-knockdown model revealed a significant reduction in the number of DILP2-positive IPCs accompanied by increased DILP2 fluorescence intensity in the axonal bundles, reproducing previously reported changes in endogenous DILP2 immunoreactivity. In addition, no detectable IgY-DILP2 immunoreactivity was observed in the midgut, providing further evidence against detectable cross-reactivity with endogenous DILP3 and DILP5 under the experimental conditions used. The antibody effectively detected endogenous DILP2 by immunofluorescence but not by Western blot analysis of fly brain extracts, potentially reflecting differences in antigen conformation and/or the low abundance of endogenous DILP2 in whole-brain samples. This study describes the generation of a polyclonal IgY antibody capable of detecting endogenous DILP2 in Drosophila tissues. The findings also suggest that the structural state of recombinant antigens may influence the generation of antibodies capable of recognizing endogenous targets. Despite the limitation of using a single hen per immunization condition, the generated IgY-DILP2 antibody represents a potentially useful tool for investigating DILP2 distribution and biology in Drosophila .