Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies.

Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. The crotoxin molecule is composed of two subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit called component-A (CA). Crotoxin exists as a mixture of several isoforms (or variants) resulting from the association of several subunit isoforms. We prepared monoclonal antibodies (MAbs) against each isolated subunit. Six anti-CA MAbs and eight anti-CB MAbs were tested for their cross-reactivities with each subunit and with other toxic and nontoxic PLA2s. Four of the six anti-CA MAbs cross-reacted with CB, whereas only one of the eight anti-CB MAbs cross-reacted with CA. Two anti-CB MAbs were found to cross-react with agkistrodotoxin, a single chain neurotoxic PLA2 purified from the venom of Agkistrodon blomhoffii brevicaudus. We determined the dissociation constants of each MAb for CA and CB isoforms and their capacities to neutralize the lethality and to inhibit the catalytic activity of crotoxin. We defined three epitopic regions on CA and four on CB, and used a schematic representation of the two subunits to characterize these epitopic regions with respect to: (1) the "toxic" and the "catalytic" sites of CB, and (2) the zone of interaction between the two subunits. We propose three-dimensional structures of the crotoxin subunits in which we localize amino acid residues that might be involved in the epitopic regions described here.

Clinical indicators of envenoming and serum levels of venom antigens in patients bitten by Bothrops lanceolatus in Martinique. Research Group on Snake Bites in Martinique.

An enzyme-linked immunosorbent assay was developed to measure venom antigen levels in the serum of 40 patients bitten by Bothrops lanceolatus. The grading system used for the severity of envenomation (grades 1 to 4, minor to major) was predominantly based on the presence of local signs. Serum venom levels increased with the grade of severity (P < 0.001, by Spearman's rank correlation test); they were 6 +/- 6 ng/mL (mean +/- SD) in clinically non-envenomed patients (grade 1, n = 3), 7.6 +/- 11.7 (n = 17), 44.3 +/- 41.8 (n = 17), and 80.3 +/- 34.1 ng/mL (n = 3) in patients diagnosed as grade 2, 3 and 4 respectively. However, venom antigens could not be detected in the serum of 54% of patients who showed clinical signs of envenomation. Most patients diagnosed as grade 2, 3 or 4 were given 20, 40 and 60 mL of a monospecific F(ab')2 antivenom, respectively. Venom concentrations > or = 15 ng/mL were observed in all patients with progressive aggravation of swelling despite the use of early antivenom therapy. No venom was detectable in blood samples taken after completion of serotherapy. All patients recovered. These results confirm the efficacy of both the clinical severity scoring system used and the therapeutic regimen.

Viper bites in France: clinical and biological evaluation; kinetics of envenomations.

1. A second inquiry was conducted in France to collect more accurate epidemiological, clinical and biological data from patients hospitalized after a viper bite, as well as treatment that they received. Fifty-seven well documented cases were classified in four grades of increasing severity defined according to the clinical signs of envenomation. 2. Local and systemic signs of envenomation appeared during the first 3 h, but the severity of the envenomation was observed to increase during the 12-24 h following bites in 50% of moderate and severe cases. One fatal case was reported. Biological analysis revealed an hyperleukocytosis in patients with moderate and severe envenomations. 3. The average length of hospitalization was of 1.7 +/- 1.3 days for patients without signs of envenomation (grade 0) or presenting a minimal envenomation (grade 1), and statistically longer, 6.2 +/- 2.9 days, for patients presenting moderate (grade 2) or severe envenomation (grade 3). 4. Levels of venom antigens in serum samples regularly collected during hospitalization were determined by a sandwich ELISA test. The serum venom levels determined during the first 4 h following the bite correlated with the severity of the envenomation when the symptoms were determined at their worst, usually 12-24 h later. In fact, concentrations higher than 20 ng ml-1 predict a moderate or severe clinical evolution. 5. The pharmacokinetics of venom antigens was also investigated during human envenomations.(ABSTRACT TRUNCATED AT 250 WORDS)

Unusual neurotoxic envenomations by Vipera aspis aspis snakes in France.

Vipera aspis aspis (V.a.a.) is the most dangerous poisonous snake in South-Eastern France. The clinical symptoms observed after V.a.a. envenomations involve mostly local signs (pain, edema) associated in the more severe cases with systemic symptoms (gastro-intestinal and cardiovascular manifestations). Since 1992, several unusual cases of moderate and severe 'neurotoxic' envenomations by V.a.a. snakes have been reported in a very localized area in South-Eastern France. Most of the human patients mainly suffered neurological signs owing to cephalic muscle paralysis. Drowsiness and dyspnea were observed for the most severe cases. Envenomed animals suffered respiratory distress and paralysis. The local signs were never as severe as observed after envenomations by vipers in other French regions. Human patients with moderate or severe clinical features received two intravenous injections of Viperfav antivenom, the first dose inducing the decrease of the neurological signs and the second reducing significantly the edema. Neurotoxic components immunologically cross-reacting with toxins from V. ammodytes ammodytes venom from Eastern Europe were detected in the blood of all patients suffering neurological symptoms after a V.a.a. bite. The protective efficacy of various antivenoms was evaluated in mice. The existence of geographical variations in the composition of V.a.a. venom emphasizes on the use of polyvalent antivenom in the treatment of viper envenomations in France.

Toxicokinetic and toxicodynamic analyses of Androctonus australis hector venom in rats: optimization of antivenom therapy.

This paper reports the simultaneous determination of toxicokinetic and toxicodynamic properties of Androctonus australis hector venom, in the absence and presence of antivenom (F(ab')(2) and Fab), in envenomed rats. After subcutaneous injection of the venom, toxins showed a complete absorption phase from the site of injection associated with a distribution into a large extravascular compartment. The injection of Fab and F(ab')(2) induced the neutralization of venom antigens in the blood compartment, as well as the redistribution of venom components from the extravascular compartment to the blood compartment. Interestingly, F(ab')(2) and Fab showed distinct efficiencies depending on their route of injection. F(ab')(2) induced a faster venom neutralization and redistribution than Fab when injected intravenously. Fab was more effective than F(ab')(2) by the intramuscular route. The hemodynamic effects of Aah venom were further investigated. Changes in mean arterial pressure and heart rate were observed in parallel with an upper airway obstruction. Fab was more effective than F(ab')(2) for preventing early symptoms of envenomation, whatever their route of administration. Intraperitoneal injection of F(ab')(2) and Fab was similar for the prevention of the delayed symptoms, even after a late administration. Fab was more effective than F(ab')(2) in the inhibition of airway resistance, independent of the route and time of administration. These results show that the treatment for scorpion stings might be improved by the intravascular injection of a mixture of Fab and F(ab')(2). If antivenom cannot be administered intravenously, Fab might be an alternative as they are more effective than F(ab')(2) when injected intramuscularly.