The giant jellyfish Nemopilema nomurai sting can cause local and systemic reactions; however, comparative analysis of the tentacle extract (TE) and nematocyst venom extract (NV), and its toxicity, mechanism, and potential intervention are still limited. This study compared venom from TE and NV for their composition, toxicity, and efficacy in vitro and in vivo used RAW264.7 cells and ICR mice. A total of 239 and 225 toxin proteins were identified in TE and NV by proteomics, respectively. Pathological analysis revealed that TE and NV caused heart and liver damage through apoptosis, necrosis, and inflammation, while TE exhibited higher toxicity ex vivo and in vivo. Biochemical markers indicated TE and NV elevated creatine kinase, lactatedehydrogenase, and aspartate aminotransferase, with the TE group showing a more significant increase. Transcriptomics and Western blotting indicated both venoms increased cytokines expression and MAPK signaling pathways. Additionally, 1 mg/kg PACOCF3 (the phospholipase A2 inhibitor) improved survival from 16.7% to 75% in mice. Our results indicate that different extraction methods impact venom activities, tentacle autolysis preserves toxin proteins and their toxicity, and PACOCF3 is a potential antidote, which establishes a good extraction method of jellyfish venom, expands our understanding of jellyfish toxicity, mechanism, and provides a promising intervention.

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 (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.