Development of a Highly Specific Immunoassay for Residual Venom Detection of the Toxic Jellyfish Nemopilema nomurai

Accurate detection of residual jellyfish venom is crucial for species identification and clinical management post-envenomation. We developed a highly specific immunoassay for <i>Nemopilema nomurai</i> 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 <i>N. nomurai</i> venom than venoms from <i>Rhopilema esculentum</i>, <i>Chrysaora quinquecirrha</i>, <i>Cyanea melanaster</i>, scorpions, or bees (<i>p</i> < 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 <i>Nemopilema nomurai</i> venom, providing a methodological basis for creating diagnostic kits for marine envenomation.