New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2.

Antivenoms are usually obtained by animal immunization with successive inoculations of increasing sublethal amounts of venom, which may impair the animal health. The high lethality of venom requires prolonged immunization plans with small amounts of venom. Thus, we propose an alternative plan that includes a pre-immunization of the animal with phospholipase A2, the main crotoxin component, which is responsible for the whole venom lethality. For comparison, three different immunization schemes were designed: high dose protocol (HDP; 0.5-27 mg of venom), low dose protocol (LDP; 0.1-7 mg of venom) and Mix protocol (MP; preimmunization 0.1-1.2 mg of crotalic PLA2, and then 4.5-8 mg of venom). Antibody titers were determined by ELISA, in blood plasma obtained from the marginal vein of the ear. The neutralizing ability of the different sera obtained by all protocols (HDS, LDS and MS) was tested against the most important pharmacological activities of whole venom: PLA2 activity, myotoxicity, thrombin like activity and lethality. MS showed the best neutralizing efficacy and at the same time, it was obtained by an immunization protocol that takes account of animal health care, since it requires low quantities of venoms in comparison to traditional protocols.

Autolysis at the disintegrin domain of patagonfibrase, a metalloproteinase from Philodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom.

Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37 degrees C, primarily producing a approximately 32.6 kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca(2+), patagonfibrase was only partially autolysed, giving rise mainly to one fragment of approximately 52.2 kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.

Evaluation of pro-inflammatory events induced by Bothrops alternatus snake venom.

Inflammation is a major local feature of envenomation by bothropic snakes being characterized by a prominent local edema, pain, and extensive swelling. There are reports demonstrating that whole Bothrops snake venoms and toxins isolated from them are able to activate macrophages functions, such as phagocytosis, production of reactive oxygen, cytokines and eicosanoids, however, little is known about the effects of Bothrops alternatus (B.a.) venom on macrophages. In this work, we evaluated the proinflammatory effects of B.a. venom with in vivo and in vitro experiments using the Raw 264.7 cell line and mouse peritoneal macrophages. We detected that B.a. venom augments cell permeability (2-fold), and cellular extravasation (mainly neutrophils), increase proinflammatory cytokines IL1 (∼300-fold), IL12 (∼200-fold), and TNFα (∼80-fold) liberation and induce the expression of enzymes related to lipid signaling, such as cPLA2α and COX-2. Additionally, using lipidomic techniques we detected that this venom produces a release of arachidonic acid (∼10 nMol/mg. Protein) and other fatty acids (16:0 and 18:1 n-9c). Although much of these findings were described in inflammatory processes induced by other bothropic venoms, here we demonstrate that B.a. venom also stimulates pro-inflammatory pathways involving lipid mediators of cell signaling. In this sense, lipidomics analysis of macrophages stimulated with B.a. venom evidenced that the main free fatty acids are implicated in the inflammatory response, and also demonstrated that this venom, is able to activate lipid metabolism even with a low content of PLA2.

Systemic pathological alterations caused by Philodryas patagoniensis colubrid snake venom in rats.

Very little is known about the systemic effects caused by Philodryas patagoniensis colubrid snake venom. In this work, this venom was tested for its ability to induce histopathological changes in rats after its intramuscular, subcutaneous or intravenous administration, by light microscopic examination of some organs (cerebellum, cerebrum, lung, liver, kidney and heart). Four rats were used for each dose of 0.23, 0.45 and 0.90 mg of venom in 0.3 ml of phosphate-buffered saline solution (pH 7.4). Aliquots of blood were withdrawn at different time intervals for enzymatic determination of alanine aminotransferase, aspartate aminotransferase and creatine kinase levels. After 2h the animals were killed by an overdose of anesthetic, and samples of kidney, heart, liver, lung, cerebrum and cerebellum were taken to microscopic examination (hematoxylin and eosin stain). Histologically, no abnormality was observed in heart tissue, in none of the administration routes of the venom used. However, histological observations showed multifocal hemorrhage in cerebellum, cerebrum and lung sections, severe peritubular capillary congestion in kidney sections and hydropic degeneration in liver sections, when venom was administrated intravenously. The subcutaneous route showed similar results to the previous one, with the exception of cerebellar hemorrhage. Intramuscularly, neither cerebral nor cerebellar hemorrhage was observed. Plasma alanine aminotransferase and aspartate aminotransferase increased levels were demonstrated, mainly when venom was administered intravenously or subcutaneously. Our results suggest that P. patagoniensis venom induces moderate histopathological changes in vital organs of rats. These changes are initiated at early stages of the envenomation and may be associated with a behavioral or functional abnormality of those organs during envenoming.

Rabbit IgG antibodies against phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom.

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 microg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d t. venom (4 microg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms.

Efficient muscle regeneration after highly haemorrhagic Bothrops alternatus venom injection.

Bothrops alternatus snake venom is particularly characterized for inducing a prominent haemorrhage and affecting hemostasis as a consequence of 43.1% of metallo-proteinases and less than 10% of PLA2 (almost all non-myotoxic phospholipases) in its venomics. In addition, myonecrosis is the major local effect in viper envenoming which might lead to permanent sequela. Then, the rebuilding of the microvasculature at the local injured site acquires significance since represents one of the pivotal stages for subsequent skeletal muscle regeneration either at morphological or functional aspects. Due to the significance played by vasculature in this process, it is important to study by histology and immunohistochemical techniques, the muscular damage and the sequence of skeletal muscle reconstruction (degree of damage, reconstitution of muscle fibres and capillaries). In this work, we injected intramuscularly 50 or 100 µg per mouse of B. alternatus venom in gastrocnemius muscles. We provided a complete description and characterization of the different stages of myogenesis after mild (50 µg) and severe (100 µg) local injury induced by B. alternatus venom toxins. The regeneration was evaluated 24 h, 3, 7, 14 and 28 days after receiving venom injection. Finally, both doses induced an extended necrosis at the site of injection where, when critical steps in the regenerative process are taking place, an efficient tissue rebuilding is achieved. B. alternatus venom is characterized by the high percentage of exclusively class P-III metalloproteinases, and by the lack of class P-I metalloproteinases in its venom composition. This could explain the effectiveness of muscle regeneration after venom injection despite the severity of the initial phase of envenoming.

P9a(Cdt-PLA2) from Crotalus durissus terrificus as good immunogen to be employed in the production of crotalic anti-PLA2 IgG.

Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all. The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins.

Haematologic alterations caused by Ipomoea carnea in experimental poisoning of guinea pig.

Ipomoea carnea subsp. fistulosa (Convolvulaceae) causes poisoning of goats, sheep and cattle in many tropical and subtropical countries. The pathophysiology of this poisoning mainly involves an abnormal glycoprotein metabolism. The aim of this study was to describe the potential toxicity of I. carnea in a guinea pig model through its effect on hematopoiesis in a time course study of 40 days. Experimental poisoning was achieved by feeding animals with "small balls" prepared with milled leaves of I. carnea mixed with commercial crushed pellets for rodents. Hematologic and biochemical parameters, bone marrow and spleencellularities, histopathologic evaluations and lectin-histochemistrywere performed during the scheduled time of the study.The treatment with "small balls" caused significant changes in the weight of spleen, a notable decrease in peripheral red blood cells, and concomitantwith morphological and histopathologicalalterationsin hematopoietic tissues. Overall, the present study suggested that 20 days ofthis treatmentcouldbe enough to develop bone marrow hypoplasia and vacuolation of white cells of spleen, blood and lymph nodes with a transient erythropoietic contribution of the splenic niche.Moreover, this work provides a cheap and simple method for detecting preclinical cases of intoxication by I. carnea in livestock.

Phospholipase A(2) enhances the endothelial cell detachment effect of a snake venom metalloproteinase in the absence of catalysis.

Microvessel disruption leading to hemorrhage stands among the most dangerous consequences of envenomings by snakes of the family Viperidae. A PIII metalloproteinase (SVMP), balteragin, purified from the venom of the snake Bothrops alternatus, displays a potent hemorrhagic effect, and a moderate myotoxicity in vivo. Previous studies described the ability of this SVMP to induce the detachment of C2C12 myoblasts in culture, without causing cytolysis. Surprisingly, a purified acidic phospholipase A2 (PLA2) from the same venom was found to increase this detaching activity of the SVMP on myoblasts. Since endothelial cells are a natural target of SVMPs in vivo, the possibility that this synergistic effect is also observed on this cell type was explored in the present work. In addition, a first approach of the mechanism of action of this effect was studied. Results clearly confirm that the acidic PLA2, despite lacking toxicity towards endothelial cells, significantly enhances the detaching effect of the SVMP even at a concentration as low as 1 µg/mL. Inhibition of enzymatic activity of the PLA2 by chemical modification with p-bromophenacyl bromide did not affect the synergistic activity, suggesting that this effect is not dependent on phospholipase enzymatic activity and may instead be the consequence of an interaction of the PLA2 with endothelial cell plasma membrane. To our knowledge, this is the first report of a synergistic action of a non toxic PLA2 in enhancing the detachment of endothelial cells induced by a metalloproteinase.

Mice plasma fibrinogen consumption by thrombin-like enzyme present in rattlesnake venom from the north-east region of Argentina.

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. "Gyroxin syndrome" in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.

Neutralisation of the pharmacological activities of Bothrops alternatus venom by anti-PLA2 IgGs.

Basic phospholipases A2 (PLA2) are toxic and induce a wide spectrum of pharmacological effects, although the acidic enzyme types are not lethal or cause low lethality. Therefore, it is challenging to elucidate the mechanism of action of acidic phospholipases. This study used the acidic non-toxic Ba SpII RP4 PLA2 from Bothrops alternatus as an antigen to develop anti-PLA2 IgG antibodies in rabbits and used in vivo assays to examine the changes in crude venom when pre-incubated with these antibodies. Using Ouchterlony and western blot analyses on B. alternatus venom, we examined the specificity and sensitivity of phospholipase A2 recognition by the specific antibodies (anti-PLA2 IgG). Neutralisation assays using a non-toxic PLA2 antigen revealed unexpected results. The (indirect) haemolytic activity of whole venom was completely inhibited, and all catalytically active phospholipases A2 were blocked. Myotoxicity and lethality were reduced when the crude venom was pre-incubated with anti-PLA2 immunoglobulins. CK levels in the skeletal muscle were significantly reduced at 6 h, and the muscular damage was more significant at this time-point compared to 3 and 12 h. When four times the LD50 was used (224 µg), half the animals treated with the venom-anti PLA2 IgG mixture survived after 48 h. All assays performed with the specific antibodies revealed that Ba SpII RP4 PLA2 had a synergistic effect on whole-venom toxicity. IgG antibodies against the venom of the Argentinean species B. alternatus represent a valuable tool for elucidation of the roles of acidic PLA2 that appear to have purely digestive roles and for further studies on immunotherapy and snake envenoming in affected areas in Argentina and Brazil.

Effect of monospecific antibodies against baltergin in myotoxicity induced by Bothrops alternatus venom from northeast of Argentina. Role of metalloproteinases in muscle damage.

Myotoxicity, one of the most relevant local manifestations in envenomation by Bothrops genus, may result from a direct action of myotoxins or be due to an indirect vascular degeneration and ischemia. Baltergin, a snake venom metalloproteinase (SVMP), isolated from Bothrops alternatus venom has been used to obtain monospecific IgG, in order to determine the relative role of toxin in myotoxicity induced by whole venom. Bothrops diporus venom, another medical relevant genus of the northeastern region of Argentina, was also studied. Anti-baltergin IgG was able to neutralize completely the hemorrhagic activity of B. alternatus venom at an antibodies:venom ratio of 30:1 (w:w). However, mice injected with B. diporus venom showed a small spot remaining even at the highest ratio of IgG:venom assayed (50:1; w:w). Specific antibodies were efficient to neutralize the myotoxicity of B. alternatus venom at ratio 30:1 (w:w) but did not neutralize the same effects in B. diporus venom. Anti-baltergin polyclonal antibodies were useful tools for revealing the central role of SVMPs in the development of myotoxicity of B. alternatus venom, as well as, helping to suggest indirectly presence of potent myotoxic phospholipases A2 (PLA2s) in B. diporus venom.

Experimental intoxication of guinea pigs with Ipomoea carnea: behavioural and neuropathological alterations.

Ipomoea carnea is a toxic plant that affects goats, with symptoms being characterised by nervous disorders and death. Swainsonine and calystegines are the principal toxic components isolated from I. carnea, which also yields lysergic acid derivatives. The aim of this study was to improve the clinical characterisation of experimental intoxication by I. carnea in guinea pigs through the evaluation of behavioural changes and to perform a thorough histopathological analysis of the affected CNS. Leaves of I. carnea were administered to guinea pigs. Open-field gait analysis and monoamine levels were measured. The poisoned animals exhibited increased vocalisation, lethargy, and a reduction in the locomotion frequency after the fourth week of intoxication, as demonstrated in the open-field test. Significant differences were observed in hind-limb gait width by the last week of intoxication. After 65 days, the guinea pigs were euthanised, necropsied, and examined using light and electron microscopy. At the end of the experiment, plasma serotonin decreased. In contrast, dopamine decreased, and noradrenaline increased in urine. Brain sections were evaluated with conventional histological methods and immunohistochemistry (IHC), as well as by transmission electron microscopy (TEM). Vacuoles were observed throughout the brain, but they were particularly prominent in the brainstem. In addition, there were PAS-negative regions, and the Nissl substance was dispersed or absent, which was confirmed with the Kluver-Barreda stain. Moderate microgliosis was observed by immunohistochemistry. In the medulla oblongata, numerous ubiquitin-positive spheroids together with neuronal degeneration were observed in the nucleus gracilis/cuneatus. Furthermore, vacuoles were observed in astrocytes, oligodendrocytes, and endothelial cells by TEM. Our results showed that the behavioural effects may have been caused by alterations in the brain in conjunction with changes in monoamine levels. This research confirms the utility of this model for studying the pathogenesis of plant-induced lysosomal storage diseases.

Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA2 from Bothrops alternatus venom on skeletal muscle (C2C12) cells.

Acute muscle damage, myonecrosis, is one of the main characteristics of envenoming by Bothrops genus. In this in vitro study we investigated the role of a metalloproteinase (baltergin) and an acidic phospholipase A2 (Ba SPII RP4) in the cytotoxicity exhibited by Bothrops alternatus venom. Baltergin metalloproteinase purified from the venom exerted a toxic effect on C2C12 myoblast cells (CC50: 583.34 µg/mL) which involved morphological alterations compatible with apoptosis/anoikis. On the contrary, the most abundant PLA2 isolated from this venom did not exhibit cytotoxicity at times and doses tested. However, when myoblasts were treated with both enzymes together, synergic activity was demonstrated. Neutralization of the venom with specific antibodies (IgG anti-baltergin and IgG anti-PLA2) confirmed this synergism.

Inflammatory effects of patagonfibrase, a metalloproteinase from Philodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom.

Patagonfibrase is a P-III class metalloproteinase isolated from the venom of Philodryas patagoniensis, a South-American, rear-fanged 'colubrid' snake responsible for accidents with clinical significance. Since local inflammatory reactions are conspicuous signs of snakebites inflicted by this species and taking into consideration that most snake venom metalloproteinases exhibit inflammatory activity, this study deals with the proinflammatory effects evoked by patagonfibrase. Herein, we demonstrate that patagonfibrase causes a time- and dose-dependent hemorrhagic edema when injected into mouse hind paws. The peak of edema occurred at 30 min after injection, and the minimum edematogenic dose was 0.021 µg. By histological analysis, the presence of moderate to marked edema and hemorrhage, and a mild inflammatory infiltrate was observed. When injected subcutaneously into the scrotal bag of mice, patagonfibrase induced cell recruitment with a significant alteration in physiological parameters of leukocyte-endothelium interaction. The presence of 1 mmol/L o-phenanthroline, which chelates metal ions, significantly inhibited the proinflammatory effects induced by patagonfibrase. Taken together, these results imply that patagonfibrase is an important contributor to local inflammation elicited by P. patagoniensis envenomation, which may pave the way for novel therapeutic strategies to treat this snakebite. Moreover, our findings demonstrate for the first time that a venom metalloproteinase from a rear-fanged snake elicits proinflammatory effects mainly mediated by its catalytic activity.

Isolation and functional characterization of a new acidic PLA(2) Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina.

An acidic protein with phospholipase A(2) activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A(2) in correspondence with Asp49 PLA(2) group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA(2) revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 microg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 microg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) "de novo amino acid sequencing", also provides more database about the small group of the non-myotoxic PLA(2)s isolated up to the present.

Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom.

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes