Sandwich ELISA using newly generated monoclonal antibodies quantifies circulating neuropeptide F in Drosophila melanogaster

Precise quantification of circulating peptide hormones is essential for understanding their physiological functions. Although the fruit fly Drosophila melanogaster is a powerful genetic model that has greatly contributed to animal physiology, measuring endogenous peptide hormone titers in the hemolymph has remained technically challenging because of its small body size. Neuropeptide F (NPF), a conserved neuropeptide produced in the central nervous system (CNS) and midgut enteroendocrine cells (EECs), has been implicated in multiple physiological processes. In particular, EEC-derived NPF is thought to function as a circulating hormone. However, endogenous hemolymph NPF titers have not been directly quantified. Here, we established a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) system for NPF using newly generated rat and mouse anti-NPF monoclonal antibodies. The ELISA system achieved a lower limit of quantification of 0.2 pM and did not detect signals in hemolymph from the NPF null mutant animals. Using this system, we observed a modest but significant increase in circulating NPF in mated females compared with virgins, consistent with the prediction from previous reports. In addition, midgut-specific knockdown of NPF reduced hemolymph NPF levels, supporting its contribution to the circulating pool. Notably, circulating NPF concentrations were in the tens of picomolar range. This level is substantially lower than the reported nanomolar receptor affinity measured in vitro, raising new questions about NPF signaling in vivo. This study provides a direct method for quantifying endogenous NPF and offers insight into peptide hormone regulation in D. melanogaster.