Development of a sensitive enzyme immunoassay for measuring taipan venom in serum

Emergency Department, Gold Coast Hospital, Queensland, Australia.
Toxicon (Impact Factor: 2.49). 03/2010; 55(8):1510-8. DOI: 10.1016/j.toxicon.2010.03.003
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The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.

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    • "Serial clinical data and laboratory results are also collected until patient discharge. Serum samples are collected when possible from all patients for measurement of venom and antivenom concentrations by specific enzyme immunoassays (Kulawickrama et al., 2010). All patient information is de-identified, coded and entered into a study database by trained personal. "
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    ABSTRACT: Intracranial haemorrhage (ICH) is a rare life-threatening consequence of venom induced consumption coagulopathy in snake-bite. It is unclear why certain patients haemorrhage. We aimed to investigate ICH in snake envenoming. Cases of venom-induced consumption coagulopathy from July 2005-June 2014 were identified from the Australian Snakebite Project, a prospective multicentre cohort of snake-bites. Cases with venom-induced consumption coagulopathy were extracted with data on the snake-bite, clinical effects, laboratory investigations, treatment and outcomes. 552 cases had venom-induced consumption coagulopathy; median age, 40y (2-87y), 417 (76%) males, 253 (46%) from brown snakes and 17 died (3%). There were 6/552 (1%) cases of ICH; median age, 71y (59-80y), three males and five from brown snakes. All received antivenom and five died. All six had a history of hypertension. Time to onset of clinical effects consistent with ICH was 8-12h in four cases, and within 3h in two. Difficult to manage hypertension and vomiting were common. One patient had a normal cerebral CT on presentation and after the onset of focal neurological effects a repeat CT showed an ICH. ICH is rare in snake-bite with only 1% of patients with coagulopathy developing one. Older age and hypertension were associated with ICH. Copyright © 2015. Published by Elsevier Ltd.
    Toxicon 05/2015; 102. DOI:10.1016/j.toxicon.2015.05.012 · 2.49 Impact Factor
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    • "The concentration of antivenom at which venom is no longer detectable can then be converted to a dose required for neutralisation. This approach appears to work well with Australian antivenoms where there is likely to be an excess of antivenom compared to venom, because the commercial antivenom is highly concentrated (O'Leary and Isbister, 2009) and the venom concentration in patients is low due to the small amount of venom delivered by elapids (Kulawickrama et al., 2010; Allen et al., 2012; Isbister et al., 2012). Therefore, venom is rarely detectable after administration of even one vial of antivenom in Australian elapid envenoming (Allen et al., 2012; Isbister et al., 2012). "
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    ABSTRACT: The measurement of free venom with enzyme immunoassay in serum of patients with snake envenoming is used to confirm snake identification and to determine if sufficient antivenom has been given. Recent studies with Russell's viper (RV; Daboia russelii) envenoming have detected free venom post-antivenom despite recovery of coagulopathy. This raises the question as to whether this assay also measures venom-antivenom (VAV) complexes. In this study we developed an assay to measure VAV complexes and investigate the binding of venom and antivenom in vitro. The assay consisted of rabbit anti-snake venom IgG attached to a microplate which binds the venom component of VAV and anti-horse IgG antibodies conjugated to horseradish peroxidase to detect the antivenom portion of VAV. A known amount of venom or toxin was incubated with increasing antivenom concentrations and VAV was detected as absorbance at 450nm and plotted against AV concentration. Pseudonaja textilis (brown snake), Notechis scutatus (tiger snake), Oxyuranus scutellatus (taipan), Tropidechis carinatus (rough-scaled snake), Pseudechis porphyriacus (red-bellied black snake) and Daboia russelii mixtures with appropriate antivenoms were assayed. Measured VAV initially increased with increasing antivenom concentration until it reached a maximum after which the VAV concentration decreased with further increasing antivenom concentrations. The VAV curves for two Australian snake venom - antivenom mixtures, H. stephensii and A. antarcticus, had broad VAV peaks with two maxima. Two fractions isolated from N. scutatus venom and Russell's viper factor X activator toxin produced similar VAV curves to their whole venoms. The antivenom concentration for which the maximum VAV occurred was linearly related to the venom concentration, and this slope or ratio was consistent with that used to define the neutralisation units for Australian antivenoms. The maximal VAV point appears to represent the antivenom concentration where every venom molecule (toxin) is attached to at least one antivenom molecule (antibody) on average and may be a useful measure of antivenom efficacy. In vivo this would mean that for a defined antivenom concentration, venom components will be eliminated and are trapped in the central compartment.
    Toxicon 11/2013; 77. DOI:10.1016/j.toxicon.2013.11.001 · 2.49 Impact Factor
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    • "Numbers indicate molecular weights. in anaesthetized rats, an effect similar to the hypotensive collapse seen in human envenoming. The potency of P. textilis venom in causing cardiovascular collapse is consistent with collapse being reported most commonly in human brown snake envenoming (Allen et al., 2012) compared to other Australian elapids (Isbister et al., 2012; Kulawickrama et al., 2010) or other snakes worldwide (Kularatne, 2003). In our animal model, cardiovascular collapse was prevented by prior administration of priming doses of the same venom (P. "
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    ABSTRACT: The Pseudonaja genus (Brown snakes) is widely distributed across Australia and bites account for significant mortality. Venom-induced consumption coagulopathy (VICC) and, less often, early cardiovascular collapse occur following envenoming by these snakes. We have previously examined possible mechanism(s) behind the early cardiovascular collapse following Papuan taipan (Oxyuranus scutellatus) envenoming. In the present study, we investigate early cardiovascular collapse in anaesthetized rats following administration of eastern brown snake (Pseudonaja textilis) venom, and prevention of this effect with prior administration of 'priming' doses (i.e. doses of venom which caused a transient hypotensive response) of venom. P. textilis venom (5-10μg/kg, i.v.) induced cardiovascular collapse in anaesthetized rats, characterized by a rapid decrease in systolic blood pressure until non recordable. Prior administration of 'priming' doses of P. textilis venom (2 and 3μg/kg) or, at least, 4-5 doses of O. scutellatus (2μg/kg, i.v.) or Daboia russelii limitis (20μg/kg, i.v.) venoms prevented cardiovascular collapse induced by P. textilis venom. Moreover, early collapse was also inhibited by prior administration of 2 discrete doses of Acanthophis rugosus venom. Prior administration of commercial polyvalent snake antivenom (500-3000 units/kg, i.v.) or heparin (300 units/kg, i.v.) also inhibited P. textilis venom-induced cardiovascular collapse. Our results indicate that P. textilis venom-induced cardiovascular collapse can be prevented by prior administration of sub lethal doses of venom from P. textilis, O. scutellatus, A. rugosus and D. russelii limitis. This suggests that sudden cardiovascular collapse following envenoming is likely to involve a common mechanism/pathway activated by different snake venoms.
    Toxicology Letters 07/2013; 221(3). DOI:10.1016/j.toxlet.2013.06.235 · 3.26 Impact Factor
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