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Comparative Evaluation of Polyclonal Antibodies in the Characterization Of Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 29 August 2018 doi:10.20944/preprints201808.0481.v1 1 Research Article 2 Comparative evaluation of polyclonal antibodies in 3 the characterization of nematocyst proteins from 4 Australian Irukandji and Chironex fleckeri species 5 Griselda Ávila-Soria1,2*, Lisa-Ann Gershwin 3,4, Kenneth D. Winkel 5, 6 and James N. Burnell2 6 7 8 1 Reef System Unit Institute of Marine Sciences and Limnology (ICML) of the National Autonomous 9 University of Mexico (UNAM), Puerto Morelos, Quintana Roo, México 10 [email protected] 11 12 2 Comparative Genomics Centre, Department of Biochemistry and Molecular Biology, School of Pharmacy 13 and Molecular Sciences, James Cook University, Townsville, Qld 4814; [email protected] 14 15 3 16 17 4 CSIRO Oceans and Atmosphere, Castray Esplanade, Hobart, Tasmania, Australia; 18 [email protected] 19 20 5 Australian Marine Stinger Advisory Services, 5/20 Hampden Road, Battery Point, Tasmania, Australia; 21 [email protected] 22 23 6 Australian Venom Research Unit (AVRU), Department of Pharmacology, The University of Melbourne. 24 25 7 Indigenous Health Equity Unit, Centre for Health Equity, School of Population and Global Health, 26 Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne; [email protected] 27 28 29 * Correspondence: [email protected]; Tel.: +52-984-235-1034 30 31 Abstract: Carukia barnesi (Cb), Malo kingi (Mk) and Chironex fleckeri (Cf) are dangerous Australian 32 box jellyfish species that provoke distinct and not well understood envenomation syndromes. 33 Specifically, Cb and Mk are small, rare and able to induce a systemic syndrome of generalised 34 muscle pain and catecholamine excess termed “Irukandji syndrome”; Cf has been widely regarded 35 as one of the most venomous organisms in the animal kingdom causing severe sting site pain 36 combined with potentially lethal cardiotoxicity. Building on past studies of major chirodropid and 37 carybdeid species venoms, this study compared the utility of various cubozoan specific antibody 38 reagents to better define the relationships between venom proteins from both exemplar Irukandji 39 species (Cb and Mk) and the archetype C. fleckeri box jellyfish. With the aid of commercial ovine 40 derived Cf-specific antivenom, mouse antibodies reactive to Cb and Mk and rabbit antibodies 41 specific to two Cf toxins (CfTX-1 and 2), as well as human sera, the cross-reactivity of jellyfish 42 species-specific polyclonal antibodies against these three cubozoan venoms was investigated. 43 Immunoblot assays revealed distinc levels of immune recognition across the three species, 44 indicating that Mk specific reagents may bind both Irukandji and Cf venoms. Irukandji venom 45 appears to be antigenic with the exception of a few proteins in the range of 43/46 kDa maybe 46 homologous to CfTX-1 and 2. The implications of such antibody binding for future antivenom 47 development require further investigation. © 2018 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 29 August 2018 doi:10.20944/preprints201808.0481.v1 48 49 Keywords: Irukandji syndrome, box jellyfish, CSL antivenom, nematocyst extracts, antigenicity; 50 human sera, human antibodies. 51 52 53 1. Introduction 54 The class Cubozoa [‘box jellyfish’] within the phylum Cnidaria consists of two orders: the 55 Carybdeida and the Chirodropida (Figure S1). Members of this phylum are characterised by the 56 presence of unique cells stinging cells (cnidae) [1] that are controlled by the nervous system; the 57 commonest of these are known as nematocysts. These are sophisticated “single-use” mechanical 58 sensor capsule-like attack devices [2], that comes in a wide variety of sizes and shapes, and typically 59 evert a barbed harpoon. The lumen of the capsule contains a complex arsenal of peptides, 60 polypeptides and bioactive lipids directed to trap, immobilize and ultimately kill prey [3]. 61 Nematocysts in Cubozoa are arranged in rings along the tentacles, and, in some species within the 62 Irukandji group, may also contain random colored warts on the exumbrella that present clusters of a 63 different type of nematocyst. The Irukandji group consists of at least 16 species ranging in size from 64 diminutive to very large [4]. The sting of these jellyfish manifests on a spectrum from mild to 65 potentially lethal depending on the particular stinging species. From smallest to largest, Irukandji 66 jellyfish include: Carukia barnesi [6], Carukia shinju [7], Malo bella [9], Malo kingi [8], Malo maxima [7], 67 Gerongia rifkinae [10], Alatina mordens [7], Morbakka fenneri [5], Keesingia gigas [9], and others. 68 69 70 The Irukandji syndrome is named after an aboriginal tribe that inhabited the Palm Cove region 71 in North Queensland, Australia, where a marine envenomation of initially unknown aetiology was 72 first reported [11,12]. The envenomation was formally named and publish by Dr Hugo Flecker in 73 1952 as occurring around Cairns, North Queensland, with C. barnesi [13] (Cb) being subsequently 74 identified as the primary causative agent for cases in that region [11,14]. The syndrome caused 75 envenomation by such species is a collection of signs and symptoms of variable severity, beginning 76 with slight sting site pain, reddening of the skin followed by a delay of ~30 minutes before the onset 77 of potentially severe systemic features, these include severe lower back pain, nausea, vomiting, 78 difficulty breathing, generalised sweating, panic/anxiety attacks, muscular spasms, abdominal 79 cramping and elevated blood pressure. In some cases cardiopulmonary decompensation leading to 80 pulmonary eodeme has been reported [4]. Affected individuals may have a combination of these 81 features, but consistently suffer musculoskeletal pain often focused around the trunk [15]. In more 82 severe cases intracerebral hemorrhage has been observed after severe systemic hypertension, 83 causing death [16], thought to be secondary to catecholamine excess. Two fatalities from Irukandji 84 syndrome were reported in 2002: one in the Whitsunday Islands, and the second off Port Douglas in 85 the tropical Great Barrier Reef [17]. According to some authors [18,19], the nematocysts obtained 86 from one of the 2002 victims were identified as a unique nematocyst type implicating M. kingi (Mk) 87 as the most likely cause of death [19]. Further, in May 2013, the deaths of two people snorkelling off 88 Coral Bay, Western Australia were potentially caused by an Irukandji jellyfish [20]. Irukandji 89 envenomations are not limited to North Queensland [14] as cases exhibiting the characteristic 90 syndrome have been reported in Broome on the West Australian coast [21,22] and as far south as 91 Victoria [23]. Irukandji syndrome is also not restricted to Australian waters as cases of the syndrome 92 are documented throughout tropical and temperate oceanic regions of the world [4,24], such as the 93 reefs off south Florida [25], the French West Indies in the Caribbean [26], Thailand [27, 28], Malaysia 94 [29] Hawaii [30], East Timor and Papua New Guinea [31]. 95 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 29 August 2018 doi:10.20944/preprints201808.0481.v1 96 97 The difficulty of studying the venom of Irukandji syndrome-causing jellyfish is compounded 98 by a number of factors. Besides being hazardous to collect, these species are rare, seasonal, often 99 small and colourless, with minimally characterized lifecycles. Consequently, since the collection of 100 the first causative species in 1961, venom analysis, and hence the development of a specific 101 treatment, has been quite slow [4]. Nevertheless recent studies have provided valuable insights of 102 the biological activity and action mechanism in Cb venom. For instance, the effect of whole Cb 103 venom on the cardiovascular tissue from humans, rats and guinea-pig (in vitro) and on anaesthetized 104 piglets (in vivo) has been reported [6], Whole Cb venom caused tachycardia and systemic pulmonary 105 hypertension triggered by a massive release of neurotransmitters, notably the catecholamines 106 adrenaline and noradrenaline [6]. These initial results have, in part, been supported by a second 107 study regarding the in vivo effects of nematocyst proteins from Cb as well as tentacle extracts devoid 108 of nematocysts tested in anaesthetized rats. The difference between the two extracts in the second 109 study was that nematocyst proteins produced cardiovascular collapse whereas the tentacle tissue 110 extract did not [32]. The experimental results of both research groups and others [33] are consistent 111 with features of catecholamine excess typical of the clinical syndrome [34]. Although these 112 experimental results explain some of the features observed in Irukandji envenomation, the 113 component responsible for the stimulation of excessive sympathetic neurotransmitter release has 114 never been purified nor has any other Cb derived toxin-related protein. 115 116 In contrast, currently the best characterized cubozoan venom, in terms of transcriptome and 117 proteome expression, as well as toxin functional characterization, is that of the multitentacled 118 Chironex fleckeri (Cf) box jellyfish [35]. Historically this species has been responsible for the most 119 severe jellyfish envenomations, and, although most are not life threatening [36], by 2004 more
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