Abstract

Accurate detection of residual jellyfish venom is crucial for species identification and clinical management post-envenomation. We developed a highly specific immunoassay for Nemopilema nomurai venom using polyclonal antibodies (titer: 1:256,000). The established i-ELISA exhibited linear detection (0–20 ng/mL) with low variability (intra-plate CV: 0.77–2.78%; inter-plate CV: 2.25–5.17%). The kit demonstrated remarkable thermal stability (<15% signal decay after 6 days at 37 °C; detectable positivity through Day 9), suggesting >1-year shelf life at 4 °C. It showed significantly higher sensitivity for N. nomurai venom than venoms from Rhopilema esculentum, Chrysaora quinquecirrha, Cyanea melanaster, scorpions, or bees (p < 0.01). Validation in murine/human skin envenomation models and serum from systemically intoxicated mice confirmed the reproducibility and stability of residual toxins. This study developed a highly sensitive, specific, reproducible, and stable i-ELISA for Nemopilema nomurai venom, providing a methodological basis for creating diagnostic kits for marine envenomation. © 2025 by the authors.