The application of immunoassay techniques,including enzyme-linked immunosorbent assay (ELISA), to snake venom research

R. D. G. Theakston. The application of immunoassay techniques, including enzyme-linked immunosorbent assay (ELISA), to snake venom research, Toxicon21, 341–352, 1983. — The development and application of immunoassay techniques in relation to snake venom research is reviewed. Enzyme linked immunosorbent assay (ELISA) is compared with radioimmunoassay, immunodiffusion, immunofluorescence, haemagglutination and immunoelectrophoresis. It is concluded that ELISA is the most versatile immunoassay technique so far applied to the field of venom research, its main advantages over other methods including relatively high levels of sensitivity and specificity, reproducibility, simplicity and ease of sample collection. It can also be readily modified into kit form and is easily adapted for use in large scale epidemiological studies and for accurate retrospective diagnosis of snake bite. None of the other assay systems considered fulfil these criteria to the same extent.ELISA is helping to advance epidemiological knowledge of snake bite, in exploring the role of active immunisation and in the compilation of accurate clinical patterns of envenoming. Other applications of the test include its use for potency screening of both new and developed commercially available antivenoms and for the detection of monoclonal antibodies which should eventually result in increased specificity of the assay system by eliminating cross reactions between venoms and antibodies of closely related species.     

Antivenom efficacy or effectiveness: The Australian experience

Despite widespread use of antivenoms, many questions remain about their effectiveness in the clinical setting. The almost universal acceptance of their value is based mainly on in vitro studies, animal studies and human observational studies. Numerous examples exist where they demonstrate clear benefit, such as consumption coagulopathy in viper envenoming, prevention of neurotoxicity in Australasian elapid bites, systemic effects in scorpion and funnel-web spider envenoming. There are also concerns about the quality and efficacy of some antivenoms. However, it is important not to confuse the efficacy of antivenom, defined as its ability to bind and neutralise venom-mediated effects under ideal conditions, and the effectiveness of antivenom, defined as its ability to reverse or prevent envenoming in human cases. There are numerous potential reasons for antivenom failure in human envenoming, of which antivenom inefficacy is only one. Other important reasons include venom-mediated effects being irreversible, antivenom being unable to reach the site of toxin-mediated injury, or the rapidity of onset of venom-mediated effects. A number of recent studies in Australia bring into question the effectiveness of some antivenoms, including snake antivenom for coagulopathy, redback spider and box jellyfish antivenoms. Despite brown snake antivenom being able to neutralise venom induced clotting in vitro, use of the antivenom in human envenoming does not appear to change the time course of coagulopathy. However, it is important that apparent antivenom ineffectiveness in specific cases is correctly interpreted and does not lead to a universal belief that antivenom is ineffective. It should rather encourage further studies to investigate the underlying pathophysiology of envenoming, the pharmacokinetics of venoms and antivenoms, and ultimately the effectiveness of antivenom based on snake type, clinical effects and timing of administration.     

Sandwich enzyme-linked immunosorbent assay for Taiwan cobra venom

Poisonous snake bite victims usually have difficulty identifying the species, and clinical manifestations alone are not reliable because of overlapping symptoms. Thus, it is important to develop a quick and reliable mean of identifying the snake responsible. We describe the development of a sandwich-ELISA method for detection of venom in biological samples and apply it to a case of snakebite to confirm the clinical diagnosis. The sandwich-ELISA takes 6 h to complete. Cobra venom antigen gave positive absorbance at about 500 pg/ml. Good linearity with R2 values over 0.99 were observed in dilution series of 1:100 ng/mL of cobra venom in calf serum and human urine. A snakebite initially thought to be Trimeresurus mucrosquamatus was proven cobra with a serum venom level up to 288 ngmL 3 h after envenoming. Sandwich-ELISA provides a rapid and accurate method for clinical identification and evaluation of toxic antigens circulating in individuals bitten by the Taiwan cobra snake.     

Efficacy of antivenom against the procoagulant effect of Australian brown snake (Pseudonaja sp.) venom: in vivo and in vitro studies.

Snake venom induced consumption coagulopathy (VICC) is a common complication of snake bite due to prothrombin activators or thrombin-like enzymes in the venom. This study aimed to determine the efficacy and dose of antivenom for treating VICC in patients envenomed by brown snakes (Pseudonaja spp.), including in vitro coagulation studies. In serial blood samples from patients with brown snake envenoming, venom and antivenom concentrations were measured using enzyme immunoassays. In vitro mixtures of brown snake venom and antivenom were used to investigate antivenom binding, neutralisation of prothrombin activity, prevention of venom-mediated clotting and effect on thrombin generation parameters using a thrombinoscope. In 27 envenomed patients the median venom concentration was 20 ng/mL (Interquartile range[IQR]:12-44 ng/mL) prior to antivenom and was not detected after antivenom administration, including 9 patients given one vial. In vitro, 200 microg/mL of antivenom bound all free venom at venom concentrations seen in patients. In vitro prothrombinase activity of the venom (using a chromogenic substrate) was not neutralised by antivenom. However, for venom concentrations seen in humans, 100 microg/mL of antivenom prevented venom clotting activity in human plasma and 479 microg/mL neutralised procoagulant venom activity measured by triggering thrombin generation. One vial of antivenom appears to be sufficient to bind and neutralise all venom in patients with severe brown snake envenoming.     

Commercial monovalent antivenoms in Australia are polyvalent

Monovalent antivenoms have a lower volume of specific antibodies that may reduce reactions but require accurate snake identification to be used. Polyvalent antivenoms are larger volume and may have a higher reaction rate. However, they avoid the problem of snake identification and may be more cost-effective to manufacture. We have previously shown cross-neutralisation of two Australian elapid venoms, tiger snake (Notechis scutatus) and brown snake (Pseudonaja textilis) venoms, by their respective monovalent antivenoms. In this study enzyme immunoassays were used to quantify the amount of monovalent antivenom (quantity of monovalent antibodies to a specific snake venom) in vials of commercially produced antivenom in Australia. All antivenoms tested appeared to be polyvalent and contain varying amounts of all five terrestrial snake monovalent antibodies based on their binding to the five representative venoms. Redback spider antivenom did not have any measurable binding affinity for any of the five snake venoms, showing that the observed binding is not due to non-specific interactions with equine protein. The antivenoms had expiry dates over a 15 year period, suggesting that the antivenoms have been mixtures for at least this time. This study cannot be used to rationalise hospital stocks of antivenom in Australia because there is no guarantee that the antivenoms will remain as mixtures. However, it would be possible for the manufacturer to reduce the number of types of snake antivenoms available in Australia to two polyvalent antivenoms which would simplify treatment of snakebite.     

A critical reappraisal of the use of enzyme-linked immunosorbent assays in the study of snake bite

The enzyme-linked immunosorbent assay (ELISA) has been the most widely used serological test in snake bite immunodiagnosis and epidemiology. The technique has been applied, however, without due consideration of the many factors which would affect an inherently sensitive test system, especially in tropical rural areas where large scale snake bite studies are usually carried out. This review discusses the effects of non-specific reactivity, cross reactivity and the quality of reagents on both the sensitivity and specificity of venom antigen and antibody detection assays. Simple laboratory modifications to optimize the assays are described. The importance of using the predictive value to assess the validity of applying the same test system in different circumstances is stressed. To fulfil its potential as the most versatile immunoassay technique in snake bite research, the test conditions of the ELISA will have to be much more stringently controlled in future.     

Cross‐Reactivity of Antivenom

Antivenom cross‐reactions have been studied using immunologic techniques, immunodiffusion, immunoelectrophoresis, immunoblotting and ELISA. Cross precipitation does not necessarily mean that there is cross protection. However, a cross protection is generally observed between closely related species. Cross‐reactivity of monovalent antivenoms and polyvalent antivenoms among various venoms are described. The results strongly suggest the presence of genus specificity in each antivenom. Cross‐reactions of antivenoms are difficult to foretell and they necessitate individual verification. Knowledge of cross‐reactivity of antivenoms is a very important tool to identify for phylogenic relationships of snake species and variation in venoms, and to be a guide for effective treatment of snake bite.     

Neutralization of two North American coral snake venoms with United States and Mexican antivenoms.

Elapid snakes throughout the world are considered very lethal, containing neurotoxic venoms that affect the nervous system. When humans are envenomated it is considered a serious medical emergency, and antivenom is the main form of treatment considered, in spite of the fact that some patients may only survive under intensive therapy treatment such as respiratory support. Coral snakes are part of the family Elapidae and envenomations by these snakes are very low (<2% of total snakebites) in most countries from southeastern United States to Argentina. In the United States, there are only two species of coral snakes of medical importance that belong to the Micrurus genera: Micrurus fulvius fulvius (Eastern coral snake) and Micrurus tener tener (Texas coral snake). In 2006, Wyeth pharmaceutical notified customers that the production of the North American coral snake antivenin (NACSA) in the US was discontinued and adequate supplies were available to meet historical needs through the end of October 2008; and therefore, it is of utmost important to consider other antivenoms as alternatives for the treatment of coral snake envenoming. One logical alternative is the coral snake antivenom, Coralmyn, produced by the Mexican company, Bioclon. In order to compare neutralization between NACSA and Coralmyn antivenoms with the North American coral snake venoms, the venom lethal doses (LD(50)) and antivenom effective doses (ED(50)) were determined in 18-20 g, female, BALB/c mice. Additionally, venom comparisons were determined through a non-reduced SDS-PAGE for M.f.fulvius, M.t.tener and the Mexican coral snake venom, Micrurus nigrocinctus nigrocinctus. Coralmyn antivenom was able to effectively neutralize three LD(50) doses of all venom from both M.t.tener and M.f.fulvius, while Wyeth antivenom only neutralized M.f.fulvius venom and was not effective in neutralizing three LD(50) doses of M.t.tener venom. Coralmyn is effective in the neutralization of both clinically important coral snake venoms in the US.     

Mass spectrophotometric evidence for P-III/P-IV metalloproteinases in the venom of the Boomslang (Dispholidus typus).

The Boomslang, Dispholidus typus, is a mid- to rear-fanged arboreal colubrid widely distributed throughout much of the African continent. Envenoming by this species is rare although deaths have been recorded. Typical symptoms associated with envenoming include diffuse intravascular coagulation (DIC) caused by fibrinogen consumption and consequent incoagulable blood together with haemorrhage into tissues such as muscle and brain; together, these procoagulant and haemorrhagic effects of the venom result in a very poor prognosis in patients who receive a large dose of venom and who are not treated with antivenom. Renal failure may also result from acute tubular necrosis resulting from pigment nephropathy. Little is known about the toxic components present in the venom; however, proteolytic activity has been reported although the proteinases involved have not been identified. In this study we provide LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) data supporting the presence of class P-III/P-IV snake venom metalloproteinases (SVMPs) in Boomslang venom. Using a polyclonal antibody raised against the P-III haemorrhagic toxin (Jararhagin) obtained from the venom of the Brazilian pit viper, Bothrops jararaca, we identified by western blot a 65 kDa protein from Boomslang venom which cross-reacted with the jararhagin antibody. A corresponding band from SDS-PAGE was subjected to tryptic digestion followed by LC/MS/MS sequence analysis of the digestion mixture. A variety of peptide sequences were identified in the digest, one of which was clearly homologous with a highly conserved region of the disintegrin-like domains of P-III/P-IV SVMPs. These data provide the first structural evidence for the presence of SVMPs in Boomslang venom; it is possible that SVMPs may also be present in the venoms of other colubrids, which cause similar symptoms in envenomed humans. In other snake venoms, most notably those of the Viperinae and Crotalinae subfamilies, many of the coagulopathic and haemorrhagic syndromes associated with systemic and local envenoming are attributed to SVMPs. The identification of a P-III/P-IV SVMP sequence in D. typus venom suggests that many of the pathological signs resulting from envenoming by this species may also be due to the presence of SVMPs in the venom. It is hoped that these results may accelerate research into colubrid venoms and may provide new insights into novel and more efficacious treatments for colubrid envenoming.     

South American rattlesnake bite (Crotalus durissus sp) without envenoming: insights on diagnosis and treatment

A South American rattlesnake bite without clinical manifestations of envenoming (termed ‘dry-bite') has not been recognized to occur by the Brazilian Ministry of Health, which recommends the administration of antivenom to all bitten patients. During 36 months of an observational study on South American rattlesnake bites in Minas Gerais, Brazil, 12% of 41 patients with fang marks at the bite-site did not present clinical or laboratory features of envenoming and had no plasma venom detected before specific serotherapy, fulfilling the criteria for the diagnosis of true ‘dry-bite'. Data from these preliminary observations suggest that these patients should be correctly diagnosed since they should not be treated with unnecessary and sometimes hazardous and expensive serotherapy.     

A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms.

A randomized blinded clinical trial was performed in 53 patients bitten by Bothrops sp. and Porthidium sp. in Antioquia and Chocó, Colombia, in order to compare the efficacy and safety of two antivenoms made of whole IgG obtained by either ammonium sulphate (monovalent anti-B. atrox) or caprylic acid (polyvalent) fractionation. Additionally, antivenoms were compared by electrophoretic and chromatographic analyses and anticomplementary activity in vitro. With a protocol of 2, 4 and 6 antivenom vials for the treatment of mild, moderate and severe envenomings, respectively, both antivenoms were equally efficient to neutralize the most relevant signs of envenoming and to clear serum venom levels in patients from the first hour and later on. Three patients with severe envenoming and initially treated with less than six vials on admission had persistent or recurrent venom antigenemia within 12-48 h. Monovalent antivenom fractionated by ammonium sulphate precipitation had higher amounts of protein aggregates and nonimmunoglobulin proteins than polyvalent antivenom fractionated by caprylic acid precipitation. Both antivenoms presented anticomplementary activity in vitro, being higher in the monovalent product. In agreement, monovalent antivenom induced a significantly higher incidence of early antivenom reactions (52%) than polyvalent antivenom (25%).     

Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms

and . Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms. Toxicon 27, 1397–1399, 1989.—Five polyvalent antivenoms (Crotalidae; Orient, North, Central and South Africa) were tested for their ability to neutralize the thrombin-like activity of snake venoms (Bitis gabonica, Agkistrodon acutus, Bothrops asper, B. atrox, Crotalus adamanteus). Considerable cross-neutralization was observed. Anti-coagulase antibodies were isolated from an antivenom by affinity chromatography using a purified enzyme from Bitis gabonica venom. These antibodies neutralized the activity of most snake venom coagulant enzymes.     

Animal experimentation in snake venom research and in vitro alternatives.

Current experimental techniques used in snake venom research (with and without the use of animals) are reviewed. The emphasis is on the reduction of the use of animals in the development of antivenoms for the clinical treatment of snakebite. Diagnostic and research techniques for the major pathologies of envenoming are described and those using animals are contrasted with non-sentient methods where possible. In particular, LD50 and ED50 assays using animals (in vivo) and fertilised eggs (in vivo, non-sentient) are compared as well as in vitro procedures (ELISA and haemolytic test) for ED50 estimations. The social context of antivenom production, supply and demand is outlined together with the consequent tension between the benefits derived and the increase in opposition to experiments on animals. Stringent regulations governing the use of animals, limited research funds and public pressure all focus the need for progress towards non-animal, or non-sentient, research methods. Some achievements are noted but success is hampered by lack of detailed knowledge of the many constituents of venom which have to be assessed as a whole rather than individually. The only way to evaluate the net pathological effect of venom is to use a living system, usually a rodent, and similarly, the efficacy of antivenoms is also measured in vivo. The pre-clinical testing of antivenoms in animals is therefore a legal requirement in many countries and is strictly monitored by government authorities. New technologies applied to the characterisation of individual venom proteins should enable novel in vitro assays to be designed thus reducing the number of animals required. In the meantime, the principles of Reduce, Refine and Replace relating to animals in research are increasingly endorsed by those working in the field and the many agencies regulating ethical and research policy.     

Toxin synergism in snake venoms

Synergism between venom toxins exists for a range of snake species. Synergism can be derived from both intermolecular interactions and supramolecular interactions between venom components, and can be the result of toxins targeting the same protein, biochemical pathway or physiological process. Few simple systematic tools and methods for determining the presence of synergism exist, but include co-administration of venom components and assessment of Accumulated Toxicity Scores. A better understanding of how to investigate synergism in snake venoms may help unravel strategies for developing novel therapies against snakebite envenoming by elucidating mechanisms for toxicity and interactions between venom components.     

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

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.     

Enzyme immunoassays in brown snake (Pseudonaja spp.) envenoming: detecting venom, antivenom and venom-antivenom complexes.

Although a commercial snake venom detection kit (SVDK) is available to distinguish between the five major snake groups in Australia, there is no assay for quantifying venom or antivenom concentrations in envenomed patients. Serum samples were obtained from patients with brown snake (Pseudonaja spp.) envenoming before and after the administration of antivenom and patients with suspected brown snake bites but no evidence of envenoming. Enzyme immunoassays (EIAs) were developed for free venom, free antivenom and the venom-antivenom complex. Standard samples measured in duplicate had a coefficient of variation of less than 10%. The EIA for venom was able to detect brown snake venom down to concentrations of 3 ng/mL. A high baseline absorbance was measured in some patients that did not change with the addition of excess antivenom to the samples. In these patients, the baseline absorbance was subtracted from all measurements to calculate the true venom concentration. The EIA for brown snake antivenom had a limit of detection of 20 microg/mL, but 50 microg/mL was used as a cut-off based on assays in patients who had not received antivenom. The EIA for venom-antivenom complexes was unable to detect these at the low venom concentrations that occurred in patients. Quantification of venom and antivenom will help to determine the dose of antivenom required to bind venom and to establish appropriate end points for antivenom treatment.     

Envenoming by viper bites in France: clinical gradation and biological quantification by ELISA.

Viper bites are frequent in France but the evaluation of the severity of envenomings and consequently patient treatment has not yet been properly evaluated. The purpose of this study was to measure venom antigens in blood and/or urine of bitten patients and to establish a quantitative relationship with clinical observations. A prospective enquiry was conducted in 1990 in France to collect epidemiological, clinical and biological data from hospitals. Urine and blood samples were tested for their content of Vipera aspis venom antigens by a sandwich enzyme linked immunosorbent assay (ELISA). One hundred and two charts were analysed, from patients presenting documented viper bites. Oedema was the prominent local feature (81 cases). Systemic signs consisted of vomiting and/or diarrhoea (22 cases), slight or severe hypotension (15 cases), shock (2 cases) and bleeding (1 case). A relationship was observed between these systemic signs and the extent of the oedema, which permitted the establishment of a grading scale. Grade 0 (no envenoming) was identified by fang marks and absence of oedema and local reaction; grade 1 (mild envenoming) by local oedema and absence of systemic symptoms; grade 2 (moderate envenoming) by regional oedema and moderate systemic symptoms; and grade 3 (severe envenoming) by extensive oedema and severe systemic symptoms. Quantification of venom antigens in blood or urine of patients by ELISA revealed a significant correlation between clinical signs of envenoming and the level of venom antigens in blood or urine. This indicated that the ELISA test is a useful and predictive tool for clinically grading viper envenomings.     

Toxoids and Antivenoms of Venomous Snakes in Taiwan

Abstract

There are in Taiwan six major venomous snakes which can inflict severe bite on human victims. They are three hemorrhagic species i.e. the Taiwan habu (Trimeresurus mucrosquamatus), the green tree viper (Trimeresurus stejnegeri), and the hundred-pace snake (Deinagkistrodon acutus); two neurotoxic species, i.e. the cobra (Naja naja atra) and the krait (Bungarus multicinctus); and the Russell's viper (Daboia r. formosensis) whose venom is both coagulopathy and neurotoxic. Our aim has been the production of highly potent antivenoms for snake-bite treatment in this country. Among individual antivenoms for Taiwan venomous snakes, only those from the pitvipers show partial cross-neutralizing capacity with venoms of other pit vipers.

As all snake venoms are quite lethal to animals, it is important to tame or detoxify the crude venom before using it on the animal to obtain antivenoms. We have demonstrated that glutaraldehyde can be used successfully not only in the detoxification of snake venoms but also improving their immunogenecity. Protocols for toxoid preparation from the crude venoms in the process of manufacturing highly potent antisera have been improved in our institute. Two bivalent equine antivenoms specific for either the combined glutaraldehyde-treated venoms of N. n. atra and B. multicinctus or those of T. mucrosquamatus and T. stejnegeri were successfully produced and proven to be effective and useful. A tetravalent equine antivenom has been prepared likewise against the four major viperid venoms in Taiwan. Recently, we also developed a process to prepare an efficient hexavalent antivenom against all the six major venomous snakes.     

Antivenom Cross-Neutralization of the Venoms of Hydrophis schistosus and Hydrophis curtus,Two Common Sea Snakes in Malaysian Waters

Sea snake envenomation is a serious occupational hazard in tropical waters. In Malaysia, the beaked sea snake (Hydrophis schistosus, formerly known as Enhydrina schistosa) and the spine-bellied sea snake (Hydrophis curtus, formerly known as Lapemis curtus or Lapemis hardwickii) are two commonly encountered species. Australian CSL sea snake antivenom is the definitive treatment for sea snake envenomation; it is unfortunately extremely costly locally and is not widely available or adequately stocked in local hospitals. This study investigated the cross-neutralizing potential of three regionally produced anti-cobra antivenoms against the venoms of Malaysian H. schistosus and H. curtus. All three antivenoms conferred paraspecific protection from sea snake venom lethality in mice, with potency increasing in the following order: Taiwan bivalent antivenom < Thai monocled cobra monovalent antivenom < Thai neuro polyvalent antivenom (NPAV). NPAV demonstrated cross-neutralizing potencies of 0.4 mg/vial for H. schistosus venom and 0.8 mg/vial for H. curtus, which translates to a dose of less than 20 vials of NPAV to neutralize an average amount of sea snake venom per bite (inferred from venom milking). The cross-neutralization activity was supported by ELISA cross-reactivity between NPAV and the venoms of H. schistosus (58.4%) and H. curtus (70.4%). These findings revealed the potential of NPAV as a second-line treatment for sea snake envenomation in the region. Further profiling of the cross-neutralization activity should address the antivenomic basis using purified toxin-based assays.     

Further studies on the mass of venom injected by Elapid snakes

Further experimental studies to determine the mass of venom injected by medically-significant Australian elapids are reported. The use of a modified enzyme immunoassay technique to measure venom injected during snake bite is presented. The feeding biting pattern of the Australian eastern brown snake (Pseudonaja textilis) is described. Using data from ten different snakes of this species, it is established that the mass of venom delivered in a first-bite is 4.69 ± 0.85 mg (mean ± S.E.) and a mean of 91% of the delivered venom is injected s.c. or into deeper tissues in a first-bite. For this species, the mass of venom delivered sequentially in a bite sequence falls to 1.32 ± 0.94 mg in the third bite in such a sequence. For the Australian rough-scaled snake (Tropidechis carinatus), the mass of venom delivered in a first feeding bite is 6.15 ± 2.23 mg, falling to a minimum of 1.92 ± 0.61 mg in the third bite of a sequence. For the Australian death adder (Acanthophis antarcticus) the mass of venom delivered in a feeding bite is 41.95 ± 16.13 mg for a first bite. Biting data is also presented for three species of the genus Pseudechis (the Australian mulga (king brown) and black snakes).