This is the first study presenting a dose–response curve for the commercially available CSL box jellyfish antivenom in a cell-based assay. The presented results clearly indicate that the effectiveness of the antivenom is dose-dependent, with higher concentrations of antivenom resulting in higher protective effects on cell survival. Similarly, a previous cell-based study using prophylactic antivenom concentrations between 0.04 and 5 units/mL on A7r5 cells (1 μg/mL of venom), found that concentrations below 5 units/mL are ineffective on cell survival . Although the latter study used a different cell type, as well as lower venom and antivenom concentrations, the benefit of higher doses of antivenom is clear.
Similarly, in vivo studies reported that venom only (30 μg/kg, i.e. for an human adult approx. 2100 μg/70 kg, thus approx. 0.81 μg/mL blood plasma) premixed with 3,000 units/kg of antivenom (ca. 10.5 vials for a 70 kg adult) was able to inhibit cardiovascular collapse in anesthetized rats, whereas concentrations of 1,000 units/kg and 600 units/kg (ca. 3.5 and 2.1 vials per 70 kg adult) failed to do so . These results slightly contradicted a previous study in which the same antivenom (3,000 units/kg) and venom dose (approx. 0.81 μg/mL) were used, yet, the majority of the anesthetized rats (60%) suffered fatal cardiovascular collapse . However, with MgSO4 as adjunct treatment all test animals survived .
The present study used far higher antivenom concentrations and thereby venom deactivation was successful (with an antivenom dose of 180 units/mL, i.e. 23.5 vials/adult), thus suggesting that the currently recommended dose for human envenomation (three vials) is probably insufficient. Recurrent criticism as to the general ability of antivenom to reduce the effects of C. fleckeri toxins may well be justified, yet not due to the serum being unable to neutralize the venom as a recent immunoaffinity assay suggested. The study was originally designed to consider differences between antibodies raised against milked, i.e. CSL-antivenom, and native venom. It has demonstrated that compared to polyclonal rabbit IgG antibodies raised against native venom, the commercially available CSL-antivenom indeed contains all the necessary antibodies to the main toxic agents in C. fleckeri venom .
However, since CSL has announced their recommendation for an initial dose of one to three vials of antivenom for human envenomation, it has been discovered that C. fleckeri venom undergoes an ontogenetic shift from invertebrate- to vertebrate-specificity with adult (as opposed to young) animals being more toxic to humans . Further, the geographic location is now known to also affect the toxicity of the venom . Consequently, the age and collection location of the specimens used to obtain the venom to raise the antibodies may affect the potency of the antivenom. Additionally, accurate quantification of venom injected into a victim is improbable, since the magnitude of envenomation depends on the degree of tentacle contact, and thus conclusions on adequate doses of antivenom for human envenomation may be inaccurate.
In the present study, the period of incubation of C. fleckeri venom with antivenom also influenced its effectiveness regarding cell survival, which suggests that antivenom is not only dose- but also time-dependent. Considering that initial beachside emergency treatment for C. fleckeri envenomation currently recommends intramuscular administration of three vials (each 20,000 units/6.25 mL), this administration route may be slow and the dose small to attenuate venom effects. In most cases of severe C. fleckeri envenomation the venom is distributed through the vascular system, thus reaching its target tissue, the heart, within minutes . In fatally envenomed victims death may occur in less than ten minutes (usually within 20 minutes) after contact with C. fleckeri tentacles [4, 19–21].
In the present study, it took more than 60 minutes to antivenom to neutralize the venom. With intramuscular injection of antivenom, delivery of the treatment would be further inhibited, since it would also have to pass through the lymphatic system in order to reach the heart and inhibit cardiovascular collapse. Conversely, if intravenous injection of antivenom could be administered as soon as possible, and life saving measures such as cardiopulmonary resuscitation could be undertaken unceasingly (as required by the victim), the antivenom would show its beneficial effects earlier. However, this partially implies that the effects of the venom are reversible (including that venom already bound to cells can be neutralized), which may contradict the currently believed pore-forming mode of action [2, 12, 22]. Finally, in the light of the rapid onset of symptoms and the fact that experimentally only prophylactic doses of antivenom were effective, the window of opportunity for antivenom administration may indeed be too short [16, 19]. However, this cannot be assumed until higher initial doses have been tried.