Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin.

Crotalus durissus terrificus (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. We have investigated the ability of commercial equine antivenom and antivenoms raised in rabbits against C. d. terrificus venom and crotoxin to neutralize the physiological and morphological changes induced by this venom and crotoxin in electrically-stimulated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations of mice. The time required to produce 50% neuromuscular blockade in the PND and EDL preparations was, respectively, 103+/-9 and 59+/-6 min for C. d. terrificus venom (10 microg/ml) and 75+/-9 and 110+/-7 min for crotoxin (10 microg/ml). The antivenoms dose-dependently inhibited this neuromuscular activity of the venom and crotoxin. At a venom:antivenom ratio of 1:3, the rabbit antivenoms were as effective as the commercial equine antivenom. The creatine kinase (CK) concentrations in the organ bath containing EDL muscle were 290 and 1020 U/l following a 120 min exposure to C. d. terrificus venom and crotoxin, respectively. All of the antivenoms neutralized the release of CK by crotoxin, but were ineffective against C. d. terrificus venom. Histological analysis of the two preparations showed that rabbit anticrotoxin antivenom protected against the myotoxic action of C. d. terrificus venom and crotoxin better than the other antivenoms. We conclude that antisera raised in rabbits are better than equine antiserum in neutralizing the neurotoxic and myotoxic activities of C. d. terrificus venom and crotoxin.     

Crotoxin: Novel activities for a classic β-neurotoxin

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric β-neurotoxin that consists of a weakly toxic basic phospholipase A₂ and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin.     

Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.     

Toxicity of Bothrops neuwiedi complex (“yarará chica”) venom from different regions of Argentina (Serpentes, Viperidae)

We report a study of toxic and enzymatic activities of Bothrops neuwiedi complex venoms collected from specimens of different regions of Argentina and a pool of these same venoms. Were determined lethal, hemorrhagic and pro-coagulant (plasma and fibrinogen) doses and the neutralization of these activities by a bivalent antivenom. The electrophoretic pattern of different regions venom was studied by SDS-PAGE. All samples exhibited lethal potencies, hemorrhagic and coagulant (plasma and fibrinogen) activities with potencies concordant with previous studies. The only conspicuous difference in the toxicological pattern of Bothrops diporus venoms was the low-thrombin-like activity found in one sample. The antivenom used in this study could neutralize all the toxic activities tested and the neutralizing potency of the antivenom was comparable for all samples. Despite the wide distribution of B. neuwiedi complex throughout Argentina and the evident morphological variation between B. diporus (B. neuwiedi complex), this study establishes a remarkably similar toxicity profile throughout its range. This is the first systematic study on the regional variation of enzymatic and toxic activities of venom from species belonging to the B. neuwiedi complex, one of the snakes of highest sanitary importance in South America and their neutralization by the type of antivenom most commonly used in the South of South America.     

Specificity of antibodies to the reconstituted crotoxin complex,from the venom of South American rattlesnake (Crotalus durissus terrificus), using enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion

Some differences between the reaction of antiserum to reconstituted crotoxin complex, to ‘native’ crotoxin and to whole Crotalus durissus terrificus (South American rattlesnake) venom were detected by enzyme-linked immunosorbent assay (ELISA) and by immunodiffusion. ELISA showed the presence of antibodies to the components of the crotoxin complex, phospholipase A₂ and crotapotin. When the antiserum was tested against North American rattlesnake venoms some species gave a positive reaction (C. horridus atricaudatus and C. basiliscus), whilst the venoms of other species were virtually negative (C. durissus totonacus, C. horridus horridus, C. viridis viridis and C. atrox). Confirmation of antigenic similarities between crotoxin and Mojave toxin (from the venom of C. scutulatus scutulatus) was obtained with ELISA and some implications of these species differences in antigen-antibody reaction are discussed. No paraspecificity was observed with heterologous snake venoms from other Crotalidae, Elapidae, Hydrophiidae or Viperidae. ELISA also showed a lack of cross-reactivity of the antiserum to heterologous purified phospholipases A₂ from Enhydrina schistosa, Naja nigricollis or Apis mellifera venoms or from porcine pancreas. The antiserum reacted to the homologous phospholipase A₂ inactivated with p-bromophenacyl bromide as well as to the detoxified crotoxin complex reconstituted with this modified phospholipase A₂. This may be useful in the raising of high titres of antibody to crotoxin.     

In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T. albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.     

Preliminary fractionation of tiger rattlesnake (Crotalus tigris) venom

S. A. Weinstein and L. A. Smith. Preliminary fractionation of tiger rattlesnake (Crotalus tigris) venom. Toxicon28, 1447–1455, 1990.—Tiger rattlesnake (Crotalus tigris) venom was fractioned by using fast protein liquid chromatography (FPLC). The crude venom had low protease activity, lacked hemolytic activity and had an i.p. ld₅₀ of 0.070 mg/kg for mice. Lethal fractions obtained by anion and cation exchange were examined for antigenic identity with crotoxin and Mojave toxin. Four toxins were obtained by anion exchange chromatography which showed immunoidentity with these toxins, and one fraction caused rear limb paresis in mice. A lethal toxin (about 10% of total venom protein) purified further with Superose-12 FPLC (molecular sieve) had an i.p. ld₅₀ of 0.050 mg/kg for mice, reacted strongly with anti-crotoxin and anti-Mojave toxin antiserum in ELISA and immunoelectrophoresis. This toxin also showed complete immunoidentity with crotoxin and Mojave toxin in immunodiffusion assays with anti-crotoxin antiserum. The results indicated the presence of crotoxin and/or Mojave toxin isoforms in this venom. Although this species has a low venom yield (average 10 mg per snake), the venom is highly toxic and contains high concentrations of several neurotoxic isotoxins.     

Snake Venom Proteomics,Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers,Ovophis convictus,Ovophis tonkinensis,and Hime Habu,Ovophis okinavensis

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40–60% of total venom proteins), followed by phospholipases A₂, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.     

Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC50s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.     

Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom

J. M. Gutiérrez, J.A. Gpené, G. Rojas and L. Cerdas. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon23, 887–893, 1985. — The polyvalent antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was tested for its capacity to neutralize proteolytic and hemorrhagic activities of ten Costa Rican crotaline venoms. In experiments with preincubation of venom and antivenom, the latter efficiently neutralized proteolytic activities of nine venoms, with ed₅₀ ranging from 50 to 300 μl antivenom/mg venom. The venom of Bothrops nummifer was neutralized less efficiently ($\text{ed}{}_{50}\text{= 760}\text{μl/mg}$). Antivenom was also very effective in neutralizing hemorrhagic activity, having its lowest neutralizing ability against the venom of B. picadoi ($\text{ed}{}_{50}\text{= 430}\text{μl/mg}$ and its highest towards the venom of B. asper (Pacific region) ($\text{ed}{}_{50}\text{= 47 μl/mg}$). There was a significant correlation between the ability of antivenom to neutralize proteolytic and hemorrhagic effects. In spite of the ability of antivenom to neutralize hemorrhage when incubated with venom prior to injection, hemorrhage was only partially neutralized when antivenom was administered i.v. at different time periods after envenomation. This suggests that the rapid development of local hemorrhage, instead of the absence of antivenom antibodies, is the explanation for the poor neutralization observed in these types of experiments.     

Immunome and venome of Bothrops jararacussu: A proteomic approach to study the molecular immunology of snake toxins

A combination of anti-bothropic and anti-crotalic sera has been reported to be more effective in neutralizing the effects of Bothrops jararacussu venom than anti-bothropic serum alone. The role of proteins from B. jararacussu venom in the horse immune response was evaluated via the analysis of cross-reactivity with homologous and heterologous sera. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis. Western blots revealed that anti-jararacussu showed higher reactivity to l-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards Snake venom serine proteases (SVSPs), PLA₂, C-type lectin and cysteine-rich proteins. Anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs. Both of these sera failed to recognize low-molecular weight proteins. Anti-crotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA₂. The cross-reactivity with anti-PLA₂ revealed the immunoreactivity of these antibodies to proteins with molecular masses in a range that is poorly recognized by other studied anti-sera. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu by may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA₂. These results also reinforce the importance of neutralizing the highly toxic proteins inclusive those with low immunogenicity in commercial antivenom production to obtain a highly protective serum against snake venoms.     

Ability of rabbit antiserum against crotapotin to neutralize the neurotoxic, myotoxic and phospholipase A2 activities of crotoxin from Crotalus durissus cascavella snake venom.

The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A(2) (PLA(2)) subunit. This PLA(2) is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA(2) alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA(2). Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA(2). In contrast, previous findings showed that antisera against crotoxin and PLA(2) from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA(2) activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.     

Cytotoxicity of Venoms and Cytotoxins from Asiatic Cobras (Naja kaouthia,Naja sumatrana,Naja atra) and Neutralization by Antivenoms from Thailand,Vietnam, and Taiwan

Envenoming by cobras (Naja spp.) often results in extensive local tissue necrosis when optimal treatment with antivenom is not available. This study investigated the cytotoxicity of venoms and purified cytotoxins from the Monocled Cobra (Naja kaouthia), Taiwan Cobra (Naja atra), and Equatorial Spitting Cobra (Naja sumatrana) in a mouse fibroblast cell line, followed by neutralization of the cytotoxicity by three regional antivenoms: the Thai Naja kaouthia monovalent antivenom (NkMAV), Vietnamese snake antivenom (SAV) and Taiwanese Neuro bivalent antivenom (NBAV). The cytotoxins of N. atra (NA-CTX) and N. sumatrana (NS-CTX) were identified as P-type cytotoxins, whereas that of N. kaouthia (NK-CTX) is S-type. All venoms and purified cytotoxins demonstrated varying concentration-dependent cytotoxicity in the following trend: highest for N. atra, followed by N. sumatrana and N. kaouthia. The antivenoms moderately neutralized the cytotoxicity of N. kaouthia venom but were weak against N. atra and N. sumatrana venom cytotoxicity. The neutralization potencies of the antivenoms against the cytotoxins were varied and generally low across NA-CTX, NS-CTX, and NK-CTX, possibly attributed to limited antigenicity of CTXs and/or different formulation of antivenom products. The study underscores the need for antivenom improvement and/or new therapies in treating local tissue toxicity caused by cobra envenomings.     

Evolution of venom antigenaemia and antivenom concentration in patients bitten by snakes in Uruguay

In this work we describe the first study carried out in Uruguay of venom antigenaemia and antivenom concentration in patients bitten by snakes. Between 50 and 70 snake bite accidents per year are caused in Uruguay by 2 species: Rhinocerophis alternatus and Bothropoides pubescens. The patients are treated with a specific polyvalent antivenom. Gaining insight on the evolution of venom antigenaemia and antivenom concentration in patients is important to improve treatment protocols. Blood samples of 29 patients were analysed to determine venom and antivenom concentrations at different times. Venom was detected in 18 of 19 samples before antivenom administration, with a mean concentration of 57 ng/mL. Most of the patients received 4 or 8 vials to neutralize the venom effects. Only one patient needed a total of 16 vials. He showed a severe envenomation and needed supplementary amounts of antivenom after the fifth day of the snake bite accident to reach normal clotting parameters. Antivenom concentrations were determined at 12 h, 24 h and 15 days after antivenom administration. It was found a faster antivenom decrease between 12 and 24 h than to 24 h to 15 days. This was explained by a different clearance mechanism in each period. In the first phase, the cause would be the neutralization of venom present in the blood whereas in the second phase it would be due to unbound antivenom elimination.     

Direct Inhibitory Effects of Carbon Monoxide on Six Venoms Containing Fibrinogenolytic Metalloproteinases

Since the introduction of antivenom administration over a century ago to treat venomous snake bite, it has been the most effective therapy for saving life and limb. However, this treatment is not always effective and not without potential life‐threatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases (SVMP) by inhibiting these Zn⁺²‐dependent enzymes directly with carbon monoxide (CO) exposure. Assessment of the fibrinogenolytic effects of venoms collected from the Arizona black rattlesnake, Northern Pacific rattlesnake, Western cottonmouth, Eastern cottonmouth, Broad‐banded copperhead and Southern copperhead on human plasmatic coagulation kinetics was performed with thrombelastography in vitro. Isolated exposure of all but one venom (Southern copperhead) to CO significantly decreased the ability of the venoms to compromise coagulation. These results demonstrated that direct inhibition of transition metal‐containing venom enzymes by yet to be elucidated mechanisms (e.g. CO, binding to Zn⁺² or displacing Zn⁺² from the catalytic site, CO binding to histidine residues) can in many instances significantly decrease fibrinogenolytic activity. This new paradigm of CO‐based inhibition of the anticoagulant effects of SVMP could potentially diminish haemostatic compromise in envenomed patients until antivenom can be administered.     

Rock squirrel (Spermophilus variegatus) blood sera affects proteolytic and hemolytic activities of rattlesnake venoms

Rock squirrels (Spermophilus variegatus) from two sites in south central New Mexico, where prairie (Crotalus viridis viridis) and western diamondback (Crotalus atrox) rattlesnakes are common predators, were assayed for inhibition of rattlesnake venom digestive and hemostatic activities. At statistically significant levels rock squirrel blood sera reduced the metalloprotease and hemolytic activity of venoms from C. v. viridis and C. atrox more than venom from an allopatric snake species, the northern Pacific rattlesnake (Crotalus oreganus). In contrast, general proteolytic activity of venom from C. oreganus was inhibited more by S. variegatus serum defenses than activity of venom from sympatric snakes. For all three venoms, incubation with squirrel sera increased the level of fibrinolysis over venom-only treatments. These results suggest that rock squirrels (S. variegatus) can defend against metalloproteases and other proteases after envenomation from at least two of five rattlesnake predators they might encounter. However, there were statistically significant differences between general proteolytic activity and fibrinolytic activity of C. v. viridis and C. atrox venom, suggesting that rock squirrels might be differentially vulnerable to these two predators. The hypothesis that prey resistance influences snake venom evolution in a predator-prey arms race is given further support by the previously cryptic variation in venoms detected when assayed against prey defenses.     

Neutralizacion de los efectos locales del veneno de Bothrops asper por un antiveneno polivalente

Neutralization of lethality, myonecrosis, hemorrhage and edema induced by Bothrops asper venom in mice was studied using the polyvalent antivenom produced in the Instituto Clodomiro Picado. The neutralizing effect (ed₅₀) on each of these toxic activities varied; the neutralization of lethal and hemorrhagic effects being more effective than the neutralization of myonecrosis and edema. With independent inoculation of venom and antivenom, antivenom was not effective in neutralizing edema-forming activity. The myonecrotic effect was only partially neutralized when serum was given i.v. immediately after envenomation; however, antivenin effectively neutralized the hemorrhagic activity. The ineffectiveness of antivenom in neutralizing edema and myonecrosis could be partially explained by the rapid development of these effects. Hence, the time interval between envenomation and antivenom administration and the route of serum administration both play an important role in the neutralization of local effects.     

Neutralization of hyaluronidase and indirect hemolytic activities of Costa Rican snake venoms by a polyvalent antivenom

J. A. Gené, M. Gómez, J. M. Gutiérrez and L. Cerdas, Neutralization of hyaluronidase and indirect hemolytic activities of Costa Rican snake venoms by polyvalent antivenom. Toxicon23, 1015–1018, 1985. — The ability of a polyvalent antivenom produced in Costa Rica to neutralize hyaluronidase and indirect hemolytic activities of Costa Rican crotalid venoms was tested. Antivenom neutralized remarkably well hyaluronidase activity of all venoms tested, with ed₅₀ doses ranging from 2 to 45 μl antivenom/mg venom. Neutralization of indirect hemolytic activity was also achieved using the antivenom, with ed₅₀ values ranging from 332 to 716μl antivenom/mg venom.     

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.     

Enzyme-linked immunosorbant assay (ELISA) of size-selected crotalid venom antigens by Wyeth's polyvalent antivenom

The binding of Antivenom (Crotalidae) Polyvalent to fractions from crude venoms of eight crotalid and one viperid snake, obtained by high performance size-exclusion chromatography, was determined with an indirect enzyme-linked immunosorbent assay (ELISA). Most of the large (greater than 30,000 mol. wt) molecular mass crotalid venom fractions were associated with high (greater than 0.7 absorbance units) ELISA values. Similarly, the medium (13,000-30,000 mol. wt) and small (less than 14,000 mol. wt) molecular mass crotalid venom fractions were coincident with moderate (0.3-0.7 absorbance units) and low (less than 0.3 absorbance units) ELISA levels. Some variability in this pattern was seen with individual venom fractions. A distinctly different pattern of ELISA values were observed with two rattlesnake venoms: the South American (Crotalus durissus terrificus) and Mojave desert (Crotalus scutulatus scutulatus) rattlesnakes. The elution profile from these venoms showed a progression of low to moderate ELISA values within the large molecular mass fractions. This pattern was followed by a decline to low ELISA values throughout the remainder of the elution profile. When saw scaled viper (Echis carinatus leucogaster) venom fractions were tested, only background ELISA values were detected with antivenom. Similarly, background ELISA values were associated with the small molecular mass fractions of all venoms tested. In addition, the elution position for the basic peptides of southern Pacific (Crotalus viridis helleri) and timber (Crotalus h. horridus) rattlesnake venoms showed minimal ELISA values. These data support the view that except for the venom of C. durissus terrificus and C. s. scutulatus, most antivenom antibodies bind large (greater than 30,000 mol. wt) venom fractions. Thus, antivenom contains minimal levels of antibodies to the basic peptides in these venoms.