Antivenom Production Research Articles

Physicochemical and biological characterization of nanovenoms, a new tool formed by silica nanoparticles and Crotalus durissus terrificus venom

Nanoparticles (NPs) are being studied due to their potential use as therapeutic and immunomodulatory tools, including their ability to transport antigens with the aim to induce a specific immune response. The production of snake antivenoms (AV) involves several inoculations of venom (V) in the presence of adjuvants (ADJ) to improve the immune response of inoculated animals, causing a decrease in its quality and shelf life. Therefore, it is interesting to develop new strategies for reduce these side effects. For that reason, associating V to NPs to replace conventional ADJ could be a useful tool for future AV production. In this work, nanovenoms (NVs) were generated by the adsorption of Crotalus durissus terrificus (Cdt) V proteins over silica NPs (SiNPs) synthesized according to the Stöber method. Microphotographies obtained under Transmission Electron Microscopy (TEM) displayed a protein crown over NPs and Fourier Transform Infrared (FT-IR) presented the expected spectra for NVs resulting from the sum of those exhibited by Cdt V and SiNPs separately. SDS PAGE and immunoblotting assays confirmed the presence of proteins over SiNPs. Furthermore, the different enzymatic activities detected demonstrated that SiNPs were capable of binding V proteins preserving its activity and therefore would keep its native structure. In the same way, the NVs conserve the potential cytotoxic effects present in the V as we observed when culturing THP-1 cells with these complexes. This evidence allows us to infer that developed NVs could be used as a new platform for the production of antisera or for immunomodulatory therapies.

Synthetic peptides to produce antivenoms against the Cys-rich toxins of arachnids.

Scorpion and spider envenomation is treated with the appropriate antivenoms, prepared as described by Césaire Auguste Phisalix and Albert Calmette in 1894. Such treatment requires the acquisition and manipulation of arachnid venoms, both very complicated procedures.Most of the toxins in the venoms of spiders and scorpions are extremely stable cysteine-rich peptide neurotoxins. Many strategies have been developed to obtain synthetic immunogens to facilitate the production of antivenoms against these toxins. For example, whole peptide toxins can be synthesized by solid-phase peptide synthesis (SPPS). Also, epitopes of the toxins can be identified and after the chemical synthesis of these peptide epitopes by SPPS, they can be coupled to protein carriers to develop efficient immunogens. Moreover, multiple antigenic peptides with a polylysine core can be designed and synthesized.This review focuses on the strategies developed to obtain synthetic immunogens for the production of antivenoms against the toxic Cys-rich peptides of scorpions and spiders.

Quantitative proteomics of Naja annulifera (sub-Saharan snouted cobra) venom and neutralization activities of two antivenoms in Africa

Envenomation by Naja annulifera (snouted cobra), a non-spitting African cobra, can result in local tissue damage and fatal paralysis but a species-specific antivenom treatment is currently lacking. In this study, we investigated the quantitative proteome of N. annulifera venom, incorporating HPLC and LC-MS/MS to elucidate the venom toxicity. The immunoreactivities and in vivo neutralization activities of two hetero-specific antivenom products (Premium Serums Pan Africa polyvalent antivenom, PANAF and VINS African polyvalent antivenom, VAPAV) against the venom were subsequently examined. N. annulifera venom comprises 10 toxin families, with three-finger toxin (3FTx) being the most abundantly expressed (~78%). Within 3FTx, cytotoxin is the most dominant form and made up three-quarter of the venom bulk (~74%), whereas alpha-neurotoxins constitute <4% of the total venom proteins. Phospholipase A2 was undetected in the venom proteome, consistent with the unusual absence of PLA2 from the venoms of cobras in the Uraeus subgenus. In ELISA, PANAF and VAPAV showed comparable immunoreactivity toward the protein antigens of N. annulifera venom. These antivenoms, despite being raised against hetero-specific venoms, were capable of cross-neutralizing the lethal effect of N. annulifera venom in mice, with PANAF being marginally more potent.

Biotechnological potential of Phospholipase D for Loxosceles antivenom development.

Loxoscelism is one of the most important forms of araneism in South America. The Health Authorities from countries with the highest incidence and longer history in registering loxoscelism cases indicate that specific antivenom should be administered during the first hours after the accident, especially in the presence or at risk of the most severe clinical outcome. Current antivenoms are based on immunoglobulins or their fragments, obtained from plasma of hyperimmunized horses. Antivenom has been produced using the same traditional techniques for more than 120 years. Although the whole composition of the spider venom remains unknown, the discovery and biotechnological production of the phospholipase D enzymes represented a milestone for the knowledge of the physiopathology of envenomation and for the introduction of new innovative tools in antivenom production. The fact that this protein is a principal toxin of the venom opens the possibility of replacing the use of whole venom as an immunogen, an attractive alternative considering the laborious techniques and low yields associated with venom extraction. This challenge warrants technological innovation to facilitate production and obtain more effective antidotes. In this review, we compile the reported studies, examining the advances in the expression and application of phospholipase D as a new immunogen and how the new biotechnological tools have introduced some degree of innovation in this field.

An Efficient Method for Endotoxin Removal from Snake Antivenoms

Parenterally administered snake antivenom immunoglobulins are the only specific treatment for envenoming by snakebites. Endotoxin removal is a necessary part of good manufacturing practice for antivenom products to avoid life-threatening consequences associated with injecting endotoxin-contaminated product. Optimization of pH is an essential factor in endotoxin purification. This study aimed to compare ultrafiltration, ion-exchange chromatography and affinity resin-based chromatography techniques at different pH values to select the depyrogenation method with the highest endotoxin removal efficiency and optimum protein/product recovery. Affinity resin-based chromatography achieved 91.2% protein recovery at acidic pH without detectable endotoxins, while ion-exchange chromatography achieved 74.42% protein recovery at pH 7.5. In contrast, ultrafiltration achieved the lowest protein recovery compared to other chromatography techniques. In addition, ultrafiltration was ineffective in removing serum albumin (~ 42–57 kDa) and low molecular weight (MW) Fc fragments (~ 24–31 kDa). In conclusion, affinity resin-based chromatography has proven to be the most effective endotoxin removal method, while ultrafiltration may not be appropriate for the removal of bacterial LPS from antivenom sera. Moreover, this study demonstrated the existence of an optimum pH for each chromatographic method for the purpose of producing sterile and endotoxin-free snake antivenoms.

Production of an alternative antivenom based on egg yolk antibodies AGAINST Crotalus durissus terrificus

Production of an alternative antivenom based on egg yolk antibodies AGAINST Crotalus durissus terrificus

Multi-epitope based immunogens applied to antivenom production

Multi-epitope based immunogens applied to antivenom production

Structure-Function Studies and Mechanism of Action of Snake Venom L-Amino Acid Oxidases.

Snake venom L-amino acid oxidases (SV-LAAOs) are the least studied venom enzymes. These enzymes catalyze the stereospecific oxidation of an L-amino acid to their corresponding α-keto acid with the liberation of hydrogen peroxide (H2O2) and ammonia (NH3). They display various pathological and physiological activities including induction of apoptosis, edema, platelet aggregation/inhibition, hemorrhagic, and anticoagulant activities. They also show antibacterial, antiviral and leishmanicidal activity and have been used as therapeutic agents in some disease conditions like cancer and anti-HIV drugs. Although the crystal structures of six SV-LAAOs are present in the Protein Data Bank (PDB), there is no single article that describes all of them in particular. To better understand their structural properties and correlate it with their function, the current work describes structure characterization, structure-based mechanism of catalysis, inhibition and substrate specificity of SV-LAAOs. Sequence analysis indicates a high sequence identity (>84%) among SV-LAAOs, comparatively lower sequence identity with Pig kidney D-amino acid oxidase (<50%) and very low sequence identity (<24%) with bacterial LAAOs, Fugal (L-lysine oxidase), and Zea mays Polyamine oxidase (PAAO). The three-dimensional structure of these enzymes are composed of three-domains, a FAD-binding domain, a substrate-binding domain and a helical domain. The sequence and structural analysis indicate that the amino acid residues in the loops vary in length and composition due to which the surface charge distribution also varies that may impart variable substrate specificity to these enzymes. The active site cavity volume and its average depth also vary in these enzymes. The inhibition of these enzymes by synthetic inhibitors will lead to the production of more potent antivenoms against snakebite envenomation.

Assessing the stability of historical and desiccated snake venoms from a medically important Ecuadorian collection

Bothrops asper and Bothrops atrox are important venomous snakes from Ecuador responsible for the most of ophidic accidents, which in the past were treated with a national polyvant antivenom. For years, the venom pools were collected and stored at room temperature in a laboratory. Taking into account the controversial ability of desiccated samples to retain their biological effects and enzymatic activities, we investigated the biochemical and toxicological properties of venoms after years of storage. The proteomic profiles of historical venoms analyzed by high-performance liquid chromatography and electrophoresis are very similar. The fresh batches of venom were more lethal than those stored for years, just as the initial and current LD50 values of these samples changed. Significant differences were showed in the myotoxic and hemorrhagic activity of some venom pools, while no significant statistical differences were found for the edema activity. The enzymatic assays revealed a variation in proteolytic activity on azocasein and phospholipase A2 activity, and low differences were reported for thrombin-like serine protease activity. The maintenance of the proteomic profile and certain toxicological activities convert this venom library in a valuable source for research purposes. Nonetheless, the significative reduction of toxicological activities, such as hemorrhagic activity not feasible using these samples for the antivenom production.

Recombinant antibodies against Iranian cobra venom as a new emerging therapy by phage display technology.

Background:The production of antivenom from immunized animals is an established treatment for snakebites; however, antibody phage display technology may have the capacity to delivery results more quickly and with a better match to local need. Naja oxiana, the Iranian cobra, is a medically important species, responsible for a significant number of deaths annually. This study was designed as proof of principle to determine whether recombinant antibodies with the capacity to neutralize cobra venom could be isolated by phage display.Methods:Toxic fractions from cobra venom were prepared by chromatography and used as targets in phage display to isolate recombinant antibodies from a human scFv library. Candidate antibodies were expressed in E. coli HB2151 and purified by IMAC chromatography. The selected clones were analyzed in in vivo and in vitro experiments. Results:Venom toxicity was contained in two fractions. Around a hundred phage clones were isolated against each fraction, those showing the best promise were G12F3 and G1F4. While all chosen clones showed low but detectable neutralizing effect against Naja oxiana venom, clone G12F3 could inhibit PLA2 activity. Conclusion:Therefore, phage display is believed to have a good potential as an approach to the development of snake antivenom.

Geographic variation of individual venom profile of Crotalus durissus snakes.

Background: South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states.Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting.Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar.Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.

Geographical variations in king cobra (Ophiophagus hannah) venom from Thailand, Malaysia, Indonesia and China: On venom lethality, antivenom immunoreactivity and in vivo neutralization

The wide distribution of king cobra (Ophiophagus hannah), a medically important venomous snake in Asia could be associated with geographical variation in the toxicity and antigenicity of the venom. This study investigated the lethality of king cobra venoms (KCV) from four geographical locales (Malaysia, Thailand, Indonesia, China), and the immunological binding as well as in vivo neutralization activities of three antivenom products (Thai Ophiophagus hannah monovalent antivenom, OHMAV; Indonesian Serum Anti Bisa Ular, SABU; Chinese Naja atra monovalent antivenom, NAMAV) toward the venoms. The Indonesian and Chinese KCV were more lethal (median lethal dose, LD50 ~0.5 μg/g) than those from Malaysia and Thailand (LD50 ~1.0 μg/g). The antivenoms, composed of F(ab)’2, were variably immunoreactive toward the KCV from all locales, with OHMAV exhibited the highest immunological binding activity. In mice, OHMAV neutralized the neurotoxic lethality of Thai KCV most effectively (normalized potency = 118 mg venom neutralized per g antivenom) followed by Malaysian, Indonesian and Chinese KCV. In comparison, the hetero-specific SABU was remarkably less potent by at least 6 to10 folds, whereas NAMAV appeared to be non-effective. The finding supports that a specific king cobra antivenom is needed for the effective treatment of king cobra envenomation in each region.

Improvement of Naja haje snake antivenom production using gamma radiation and a biotechnological technique

This study aimed to improve antivenom production using gamma radiation and calcium phosphate nanoparticles (CPN) as adjuvant. Toxicological and immunological properties of the Naja haje venom before and after exposure to radiation were estimated. The immune response of immunized rabbits was evaluated. Toxicity of irradiated venom was reduced six times as compared to native venom. Moreover, no change in antigenic reactivity between native and irradiated venoms. Serum titer produced with irradiated venom loaded on CPN showed highest titer and neutralization capacity as compared to other sera. Thus, irradiation and nanoparticles adjuvant could be a new approach for snake antivenom production.

Bradykinin-Potentiating Factors of Venom from Iranian Medically Important Scorpions.

The venom of animals, including snakes, scorpions, and spiders is a complex combination of proteins, peptides, and other biomolecules as well as some minerals. Among the biomolecules of some animal&amp;rsquo;s venom, small peptides that lack disulfide bands known as Non-Disulfide Bridge Peptides (NDBPs) potentiate the bradykinin by preventing the conversion of angiotensin 1 to angiotensin 2 using the mechanism of inhibiting the Angiotensin-Converting Enzyme activity and finally reducing the blood pressure in the victims. This feature of the NDBPs of animal&amp;rsquo;s venom is suggested as the potential of biological drugs. This study aimed to isolate venom components of three species of Iranian medically important scorpions and study the bradykinin potentiating effect of them. The scorpion specimens were collected from the venomous animals and antivenom production department of Razi Vaccine and Serum Research Institute, Karaj, Iran. Moreover, venom extraction was performed by electrical shock (5 volts). The obtained liquid venom of three species specimens was frozen and lyophilized immediately and then preserved in a cool and dried place. The isolation of the venom components for each scorpion was carried out using high-performance liquid chromatography. The obtained ranges of venom fractions (zones) were tested on isolated tissues of guinea-pig ileum and rat uterus using organ bath instrumentation in several replicates. The bioassays resulted in the peptides, including Z1 and Z2 regions in the venom fractionsof the Hottentotta saulcyi, Z2 in Odontobuthus doriae, as well as Z2 and Z3 in Mesobuthus eupeus demonstrated bradykinin potentiating effect. It is concluded that Bradykinin Potentiating Factors were traceable in the venom of all three scorpion species. Therefore, these venoms have the therapeutic potential to exploit biological-based drugs.

A Combined Strategy to Improve the Development of a Coral Antivenom Against Micrurus spp.

Accidents involving Micrurus snakes are not the most common ones but are noteworthy due to their severity. Victims envenomed by Micrurus snakes are at high risk of death and therefore must be treated with coral antivenom. In Brazil, the immunization mixture used to fabricate coral antivenom contains Micrurus frontalis and Micrurus corallinus venoms, which are difficult to be obtained in adequate amounts. Different approaches to solve the venom limitation problem have been attempted, including the use of synthetic and recombinant antigens as substitutes. The present work proposes a combined immunization protocol, using priming doses of M. frontalis venom and booster doses of synthetic B-cell epitopes derived from M. corallinus toxins (four three-finger toxins-3FTX; and one phospholipase A2-PLA2) to obtain coral antivenom in a rabbit model. Immunized animals elicited a humoral response against both M. frontalis and M. corallinus venoms, as detected by sera reactivity in ELISA and Western Blot. Relevant cross-reactivity of the obtained sera with other Micrurus species (Micrurus altirostris, Micrurus lemniscatus, Micrurus spixii, Micrurus surinamensis) venoms was also observed. The elicited antibodies were able to neutralize PLA2 activity of both M. frontalis and M. corallinus venoms. In vivo, immunized rabbit sera completely protected mice from a challenge with 1.5 median lethal dose (LD50) of M. corallinus venom and 50% of mice challenged with 1.5 LD50 of M. frontalis venom. These results show that this combined protocol may be a suitable alternative to reduce the amount of venom used in coral antivenom production in Brazil.

Morphological alterations caused by manual venom extraction on the main venom gland of Bothrops asper and Crotalus simus snakes (Serpentes: Viperidae): Long-term implications for antivenom production

The only scientifically validated treatment for snakebite envenomation is the administration of antivenoms. For their production, small quantities of snake venom are injected in animals to elicit a specific antibody response. Snakes are kept in captivity, and their venom is regularly extracted to assure antivenom access. It has already been reported that the pressure exerted upon the venom gland during this extraction can cause tissue damage and fibrosis, leading to a decrease in the venom yield. We described the histopathology of venom glands for B. asper and C. simus snakes used for antivenom production. Based on these reported tissue abnormalities, we quantify the tissue injury by a generated damage-SCORE and fibrosis. A variety of histopathological damages were found such as fibrosis, edema, necrosis, hemorrhage, and formation of anomalous structures, especially in C. simus, which is more prone to suffer severe damage. The level and severity of the damage depend on the frequency and the number of venom extractions. Furthermore, we design an experimental intensive venom extraction scheme with which we could confirm the causality of these effects. In addition to the histopathological damages, the LD50 and biochemical venom composition were also affected giving experimental evidence that the venom extraction not only causes tissue damage but also affects the composition stability and toxicity of the venom. In order to produce quality and effective antivenoms, an improvement of the management of snake collections could be established, such as rotation groups to assure the quality of the venom yielded.

Novel three-finger toxins from Micrurus dumerilii and Micrurus mipartitus coral snake venoms: Phylogenetic relationships and characterization of Clarkitoxin-I-Mdum

Micrurus mipartitus and M. dumerilii are the most medically important coral snakes in Colombia. Proteomic characterization of their venoms has previously shown that proteins of the three-finger toxin (3FTx) family are abundant components, especially in M. mipartitus (61%) and to a lesser extent in M. dumerilii (28%). In order to increase knowledge on these toxins, in this work a major 3FTx of M. dumerilii venom (8% of the venom proteins), named Clarkitoxin-I-Mdum, was isolated and characterized. Its amino acid sequence comprises 66 residues, with an isotope-averaged molecular mass of 7537 ± 2 Da and a theoretical pI of 9.36, presenting the conserved pattern of eight cysteines that classifies it as a short-chain (type I) 3FTx. Clarkitoxin-I-Mdum was not lethal to mice by intravenous or intracerebroventricular route and was not cytolytic to myogenic cells in vitro. On the other hand, five coding sequences for 3FTxs were obtained from the venom gland of M. mipartitus. These novel toxin sequences were named Mm3FTx-01 to Mm3FTx-05, all of them also presenting the eight conserved cysteines of short-chain 3FTxs. Phylogenetic analysis revealed high variability of 3FTxs from Micrurus, and ELISA using antibodies raised to the major 3FTxs from M. mipartitus and M. dumerilii confirmed their immunochemical divergence. These results highlight the relevance of performing further studies aiming at a deeper understanding of the functional and antigenic relationships among specific Micrurus toxins, with important implications for the production of antivenoms.

Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency

Accidents with venomous snakes are a major health hazard in tropical countries. Bothrops genus is responsible for almost 80% of snakebites in Brazil. Immunotherapy is the only approved specific treatment against snake toxins and the production of therapeutic antivenoms requires quality control tests to determine their neutralizing potency. Currently, these controls are performed by in vivo lethality neutralization, however, the inhibition of particular events produced by bothropic venoms such as coagulopathy, hemorrhage, edema or cytotoxic effects are also required. The aim of this work is to develop an in vitro alternative assay for antivenom pre-clinical evaluation. In this sense, we designed a cell viability assay using different amounts (0.2–10 μL/well) of low and high potency anti-bothropic sera, previously classified by the traditional in vivo test, for assessing the antivenom capacity to protect the cells against B. jararaca venom cytotoxicity (5xEC50 = 58.95 μg/mL). We found that high potency sera are more effective in neutralizing B. jararaca venom cytotoxicity when compared to low potency sera, which is in accordance to their pre-determined in vivo potency. Considering sera in vitro inhibitory concentration able to prevent 50% cell death (IC50) and their known in vivo potency, a cut-off point was determined to discriminate low and high potency sera. Our data provide insights for the development of an in vitro method which can determine the anti-bothropic antivenom potency during its production.

Cross-Reactivity and Neutralization Capacity of Polyspecific Antivenom Produced by Razi Institute against Three Species of Buthidea Family Scorpions

Abstract :Scorpion sting is a significant health problem in southern provinces of Iran. Many thousands of people are stung by different species of scorpions annually. More than 60 scorpion species have been reported to be present in Iran. However razi Institute manufactures polyvalent antivenom against only six species of scorpions, excluding Buthacus macrocentrus , Apistobuthus susanae and Vachoniolus iranus, wide spread scorpion species in south region of Iran. In this research, after collecting scorpions (at night),milking by elctroshock method, the venom of its lyophilized and the LD50 was determined. The fractions were then separated by gel chromatography and HPLC. Using SDS page electrophoresis, the fractional molecular weight was determined. At the end, potency test of these scorpion venoms was carried out in the vicinity of the antiserum produced by the Razi Institute on the animal.In the present work we tried to investigate the cross reactivity of present antivenom against these excluded scorpion species. The antisera production of the Razi Institute was able to neutralize the 33 LD50 of Apistobuthus susanae venom. This antivenom could neutralize Buthacus macrocentrus as well as Vachoniolus iranus scorpion venoms by 41 LD50 and 15 LD50 respectively . According to the World Health Organization (WHO) , antiserums should be made locally and against animal species.Therefore based on the results obtained in the present study it can be concluded that although the venom of these scorpion species are not included in the antivenom production but the present polyvalent antivenom is able to be used in patients stung by these 3 species of scopions too. The powerity of anti-serum neutralizing against the venom of other species of scorpions is based on the constructional similarity and cross-reactivity of different species of venom scorpions

Clinical implications of convergent procoagulant toxicity and differential antivenom efficacy in Australian elapid snake venoms

Australian elapid snakes are some of the most venomous snakes in the world and are unique among venomous snakes in having mutated forms of the blood clotting factor X in an activated form (FXa) as a key venom component. In human bite victims, an overdose of this activated clotting enzyme results in the systemic consumption of fibrinogen due to the large amounts of endogenous thrombin generated by the conversion of prothrombin to thrombin by venom FXa. Within Australian elapids, such procoagulant venom is currently known from the tiger snake clade (Hoplocephalus, Notechis, Paroplocephalus, and Tropidechis species), brown/taipan (Oxyuranus and Pseudonaja species) clade, and the red-bellied black snake Pseudechis porphyriacus. We used a STA-R Max coagulation analyser and TEG5000 thromboelastographers to test 47 Australian elapid venoms from 19 genera against human plasma in vitro. In addition to activity being confirmed in the two clades above, FXa-driven potent procoagulant activity was found in four additional genera (Cryptophis, Demansia, Hemiaspis, and Suta). Ontogenetic changes in procoagulant function was also identified as a feature of Suta punctata venom. Phylogenetic analysis of FX sequences confirmed that snake venom FXa toxins evolved only once, that the potency of these toxins against human plasma has increased in a stepwise fashion, and that multiple convergent amplifications of procoagulant activity within Australian elapid snakes have occurred. Cofactor dependence tests revealed all procoagulant venoms in our study, except those of the tiger snake clade, to be highly calcium-dependent, whereas phospholipid dependence was less of a feature but still displayed significant variation between venoms. Antivenom testing using CSL Tiger Snake Antivenom showed broad but differential cross-reactivity against procoagulant venoms, with P. porphyriacus and S. punctata extremely well neutralised but with Cryptophis, Demansia, and Hemiaspis less well-neutralised. The relative variation was not in accordance to genetic relatedness of the species used in antivenom production (Notechis scutatus), which underscores a fundamental principle that the rapid evolution characteristic of venoms results in organismal phylogeny being a poor predictor of antivenom efficacy. Our results have direct and immediate implications for the design of clinical management plans in the event of snakebite by such lesser known Australian elapid snake species that have been revealed in this study to be as potent as the better studied, and proven lethal, species.