G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse pl... more G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351-363, 1994.--A simple methodology for hyperimmune horse plasma fractionation, based on caprylic acid precipitation, is described. Optimal conditions for fractionation were studied; the method gives best results when concentrated caprylic acid was added to plasma, whose pH had been adjusted to 5.8, until a final caprylic acid concentration of 5% was reached. The mixture was vigorously stirred during caprylic acid addition and then for 60 min; afterwards the mixture was filtered. Non-immunoglobulin proteins precipitated in these conditions, whereas a highly enriched immunoglobulin preparation was obtained in the filtrate, which was then dialysed to remove caprylic acid before the addition of NaC1 and phenol. Thus, antivenom was produced after a single precipitation step followed by dialysis. In order to compare this methodology with that based on ammonium sulfate fractionation, a sample of hyperimmune plasma was divided into two aliquots which were fractionated in parallel by both methods. It was found that caprylic acid-fractionated antivenom was superior in terms of yield, production time, albumin/globulin ratio, turbidity, protein aggregates, electrophoretic pattern and neutralizing potency against several activities of Bothrops asper venom. Owing to its efficacy and simplicity, this method could be of great value in antivenom and antitoxin production laboratories.
A comparative study was performed on the venoms of adult specimens of the neotropical rattlesnake... more A comparative study was performed on the venoms of adult specimens of the neotropical rattlesnake, Crotalus durissus, from Guatemala, Costa Rica, Venezuela and Brazil, together with the venom of newborn specimens of C. d. durissus from Costa Rica. Venoms from Brazil (C. d. terrificus) and from newborn specimens of C. d. durissus presented an electrophoretic pattern characterized by the predominance of bands with molecular mass of 36 and 15 kDa, whereas those of adult specimens of C. d. durissus from Guatemala and Costa Rica, and C. d. cumanensis from Venezuela, showed a conspicuous band of 62 kDa, and additional bands of 36, 29 and 15 kDa. Moreover, venoms from C. d. terrificus and C. d. cumanensis showed a prominent band of < 10 kDa that probably corresponds to crotamine, since a…
Bothrops asper is responsible for approximately half of the snakebite envenomations in Central Am... more Bothrops asper is responsible for approximately half of the snakebite envenomations in Central America. Despite its medical relevance, only the venom of Costa Rican populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. Venom of B. asper from Guatemala was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis, and its basic pharmacological activities were investigated with standard laboratory assays. Venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating and phospholipase A(2) activities, showing a similar toxicological profile to the one previously described for B. asper from Costa Rica. In addition, polyvalent antivenoms produced in Mexico and Costa Rica, and currently used in Guatemala, were tested for their ability to neutralize venom's toxic activities. Both antivenoms were effective against all effects studied, although the Costa Rican product showed higher potency against most activities tested and higher antibody titer against venom components, as determined by enzyme immunoassay. It is suggested that different dosage regimes should be considered when using these antivenoms in B. asper envenomations in Guatemala.
Intravenous administration of antivenoms is associated with early adverse reactions in a number o... more Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4°C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.
J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic pho... more J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum. Taxicon 25, 1244Taxicon 25, -1248Taxicon 25, , 1987 . -The myotoxin from B. asper snake venom inhibited Ca-ATPase activity of rabbit sareoplasmie retieulum after incubation in vitro . Inhibition was non-competitive and albumin enhanced the effect of the toxin . Furthermore, B. riper myotoxin hydrolyzed sarcoplasmic reticulum phospholipids and induced a dose-dependent release of horseradish perolddase that had been trapped in sarcoplasmic reticulum vesicles . Binding studies indicated that myotoxin does not bind to any particular protein of this membrane, suggesting that the toxin might interact with phospholipids. Inhibition of Ca-ATPase is probably a consequence of an alteration in sarcoplasmic reticulum phospholipids .
The mortality rate due to snakebite envenomation in Costa Rica was estimated from 1952 to 1993. T... more The mortality rate due to snakebite envenomation in Costa Rica was estimated from 1952 to 1993. The highest mortality was observed during the 1950s and 1960s, with the highest rate of 4.83 per 100,000 population in 1953. In contrast, a rate of 0.2 per 100,000 population per year was estimated from 1990 to 1993. The most conspicuous decline in mortality occurred after 1970. The highest mortality rates were observed in the provinces of Limón and Puntarenas, especially in regions where tropical rain forests had been transformed into agricultural fields. The lowest mortality was in the province of Guanacaste, where tropical dry forest predominates and Bothrops asper (terciopelo), the most important poisonous snake in the country, is not abundant. The majority of fatalities occurred in the age groups from 10 to 19 years old. Males were more affected than females in a ratio of 3.6:1. Before 1980 most fatal cases did not receive medical attention in hospitals, whereas after 1980 the majority of cases with fatal outcome were attended in hospitals.
of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1... more of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1993.-Twenty batches of polyvalent antivenom produced at the Instituto Clodomiro Picado were analyzed for turbidity, both before and after freezing-thawing and lyophilization . Eight batches became turbid upon freezing-thawing, and this change correlated with high levels of cholesterol, triglycerides and lipoproteins, especially ß-lipoprotein. Since normal horse serum does not become turbid after freezing-thawing, despite the fact that it has high lipoprotein levels, the possibility was raised that phenol, used as a preservative during serum fractionation, might affect lipoproteins, inducing the appearance of turbidity after freezing-thawing . This hypothesis was tested by fractionating a sample of hyperimmune serum either without phenol or using two different phenol concentrations (0.1 g/dl and 0.25 g/dl). Results showed that, although the three samples had the same cholesterol and triglyceride levels before fractionation, only the one having 0.25 g/dl phenol became turbid upon freezing-thawing, containing a diffuse lipoprotein band on electrophoresis. This finding suggests that turbidity in equine antivenoms depends on the interaction of at least three factors: (a) freezing, (b) high initial cholesterol and lipoprotein concentration in the serum, and (c) addition of phenol during fractionation of serum.
The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops a... more The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa2EDTA than when receiving booster injections of venom alone, although they showed a similar antivenom response. Moreover, antivenoms produced from plasmas of horses that received booster injections of either venom alone or venom plus CaNa2EDTA had similar neutralizing activity against lethal, hemorrhagic and coagulant effects induced by B. asper venom. The similar antibody response was corroborated by Western blotting using crude venom and by an ELISA that estimates anti-myotoxin titer. It is concluded that the chelating agent CaNa2EDTA reduces the extent of local tissue damage induced by B. asper venom, without affecting the immune response of horses against pharmacologically-relevant venom components.
A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize let... more A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize lethal and myotoxic activities of Micrurus nigrocinctus venom. Both antivenoms were adjusted to a similar neutralizing potency in experiments where venom and antivenoms were preincubated prior to injection. No significant differences were observed between IgG and F(abЈ) 2 antivenoms concerning neutralization of lethal effect in rescue experiments, i.e., when antivenom was administered intravenously after envenomation. However, F(abЈ) 2 antivenom was more effective in prolonging the time of death when subneutralizing doses were administered immediately after venom injection. Both products partially reversed the binding of M. nigrocinctus ␣-neurotoxins to acetylcholine receptor in vitro. The IgG and F(abЈ) 2 antivenoms effectively neutralized venom-induced myotoxicity when administered intravenously immediately after envenomation, although neutralization was poor if antivenom injections were delayed. Intramuscular injection of venom promoted diffusion of antivenom antibodies throughout muscle tissue, and F(abЈ) 2 diffused to a higher extent than IgG molecules. Thus, despite the observation that F(abЈ) 2 antivenom was more effective than IgG antivenom in prolonging the time of death when subneutralizing doses were administered immediately after envenomation, no major differences were observed in antivenom neutralization of lethal and myotoxic effects or in their capacity to reverse neurotoxin binding to the acetylcholine receptor.
A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutral... more A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutralize lethal, hemorrhagic and coagulant activities of the venoms of 16 species of Central and South American snakes of the subfamily Crotalinae. Neutralization of lethality was studied by two dierent methods routinely used in the quality control of antivenoms at Instituto Butantan (IB) and Instituto Clodomiro Picado (ICP). Both antivenoms neutralized the majority of the venoms studied, but the values of eective doses 50% (ED 50 ) diered markedly depending on the method used. In general, higher potencies were obtained with the method of ICP, where a challenge dose corresponding to 4 LD 50 s is used, than with the method of IB, where a challenge dose of 5 LD 50 s is employed. All venoms induced hemorrhagic activity in the mouse skin test, which was eectively neutralized by the two antivenoms. All venoms, except those of Porthidium nasutum and Bothriechis lateralis, induced coagulation of human plasma in vitro and both antivenoms were eective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive
Transactions of The Royal Society of Tropical Medicine and Hygiene, 2005
A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a... more A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a mixture of the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the three most medically important snakes in sub-Saharan Africa. The antivenom is a whole IgG preparation, obtained by caprylic acid precipitation of non-IgG plasma proteins. The antivenom effectively neutralizes the most important toxic activities of the three venoms used in the immunization in standard assays involving preincubation of venom and antivenom before testing. This antivenom compares favourably with other antivenoms designed for use in Africa with respect to neutralization of the toxins present in the venom of E. ocellatus. Caprylic acid fractionation of horse hyperimmune plasma is a simple, convenient and cheap protocol for the manufacture of high quality whole IgG antivenoms. It constitutes a potentially valuable technology for the alleviation of the critical shortage of antivenom in Africa.
The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom... more The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was studied during a twelve-month period. The following parameters were evaluated: neutralizing potency against lethal activity of Bothrops asper venom; protein and phenol concentrations; pH; turbidity; safety; and sterility. Analyses were performed each month on different samples of a batch, stored at 4, 23, 30 and 37 degrees C. No significant (P greater than 0.1) variations occurred in potency, protein and phenol concentrations, pH, sterility or safety, at any of the storage temperatures during the study period. However, visual inspection revealed a moderate increase in turbidity of the samples stored at 23, 30 and 37 degrees C, at nine, four and three months, respectively. Culture of samples excluded the possibility of microbial contamination of the product leading to turbidity. Chromatographic and electrophoretic analyses demonstrated that turbidity was caused by the formation of heterogeneous protein aggregates of high molecular weight. Present results support the conclusion that, although storage temperature (up to 37 degrees C for twelve months) does not alter antivenom potency, it significantly influences the formation of protein aggregates. This phenomenon can be prevented by recommending the storage of antivenom at refrigeration temperature.
A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ec... more A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ecacy in the neutralization of several toxic and enzymatic activities of the venoms of B. lanceolatus, B. atrox and B. asper. When tested by the i.p. route in mice, B. lanceolatus venom had an LD 50 of 12.8 mg/g. In addition, it induced local tissue damage (hemorrhage, edema and myotoxicity) and showed indirect hemolytic activity, but was devoid of coagulant eect on human plasma in vitro and of de®brinating activity in mice. Antivenom was fully eective in the neutralization of lethal, hemorrhagic, edema-forming, myotoxic and indirect hemolytic eects of B. lanceolatus venom in assays involving preincubation of venom and antivenom. When tested against the venoms of B. asper and B. atrox, the antivenom completely neutralized the lethal, hemorrhagic, myotoxic and indirect hemolytic eects, and was partially eective in neutralizing edema-forming activity. In contrast, the antivenom was ineective in the neutralization of in vitro coagulant and in vivo de®brinating eects induced by these two venoms. #
Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health ha... more Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health hazard in Perú, and the intravenous administration of equine-derived antivenoms represents the only scientifically validated treatment. This study presents a preclinical assessment of the efficacy of two whole IgG antivenoms, prepared in Perú and Costa Rica, to neutralize the most relevant toxic effects induced by the venoms of Bothrops atrox, B. brazili, B. barnetti and B. pictus from Perú. Peruvian antivenom is produced by immunizing horses with Bothrops sp. venoms from this country, whereas the production of Costa Rican antivenom involves immunization with venoms from Central American snakes. The neutralization of lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating activities was evaluated in assays involving incubation of venom and antivenom prior to testing. Both antivenoms were effective in the neutralization of these effects, with quantitative variations in the values of effective dose 50% depending on the effects being studied. Peruvian antivenom was more effective in the neutralization of lethality induced by B. atrox and B. barnetti venoms. However, Peruvian antivenom failed to neutralize coagulant activity of B. barnetti venom and edema-forming activity of B. brazili venom, whereas neutralization was achieved by Costa Rican antivenom. It is concluded that an extensive immunological cross-reactivity exists between Bothrops sp. venoms from Perú and Costa Rica, and that both antivenoms are effective in the neutralization of these four venoms in a rodent model of envenoming.
G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse pl... more G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351-363, 1994.--A simple methodology for hyperimmune horse plasma fractionation, based on caprylic acid precipitation, is described. Optimal conditions for fractionation were studied; the method gives best results when concentrated caprylic acid was added to plasma, whose pH had been adjusted to 5.8, until a final caprylic acid concentration of 5% was reached. The mixture was vigorously stirred during caprylic acid addition and then for 60 min; afterwards the mixture was filtered. Non-immunoglobulin proteins precipitated in these conditions, whereas a highly enriched immunoglobulin preparation was obtained in the filtrate, which was then dialysed to remove caprylic acid before the addition of NaC1 and phenol. Thus, antivenom was produced after a single precipitation step followed by dialysis. In order to compare this methodology with that based on ammonium sulfate fractionation, a sample of hyperimmune plasma was divided into two aliquots which were fractionated in parallel by both methods. It was found that caprylic acid-fractionated antivenom was superior in terms of yield, production time, albumin/globulin ratio, turbidity, protein aggregates, electrophoretic pattern and neutralizing potency against several activities of Bothrops asper venom. Owing to its efficacy and simplicity, this method could be of great value in antivenom and antitoxin production laboratories.
A comparative study was performed on the venoms of adult specimens of the neotropical rattlesnake... more A comparative study was performed on the venoms of adult specimens of the neotropical rattlesnake, Crotalus durissus, from Guatemala, Costa Rica, Venezuela and Brazil, together with the venom of newborn specimens of C. d. durissus from Costa Rica. Venoms from Brazil (C. d. terrificus) and from newborn specimens of C. d. durissus presented an electrophoretic pattern characterized by the predominance of bands with molecular mass of 36 and 15 kDa, whereas those of adult specimens of C. d. durissus from Guatemala and Costa Rica, and C. d. cumanensis from Venezuela, showed a conspicuous band of 62 kDa, and additional bands of 36, 29 and 15 kDa. Moreover, venoms from C. d. terrificus and C. d. cumanensis showed a prominent band of < 10 kDa that probably corresponds to crotamine, since a…
Bothrops asper is responsible for approximately half of the snakebite envenomations in Central Am... more Bothrops asper is responsible for approximately half of the snakebite envenomations in Central America. Despite its medical relevance, only the venom of Costa Rican populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. Venom of B. asper from Guatemala was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis, and its basic pharmacological activities were investigated with standard laboratory assays. Venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating and phospholipase A(2) activities, showing a similar toxicological profile to the one previously described for B. asper from Costa Rica. In addition, polyvalent antivenoms produced in Mexico and Costa Rica, and currently used in Guatemala, were tested for their ability to neutralize venom's toxic activities. Both antivenoms were effective against all effects studied, although the Costa Rican product showed higher potency against most activities tested and higher antibody titer against venom components, as determined by enzyme immunoassay. It is suggested that different dosage regimes should be considered when using these antivenoms in B. asper envenomations in Guatemala.
Intravenous administration of antivenoms is associated with early adverse reactions in a number o... more Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4°C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.
J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic pho... more J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum. Taxicon 25, 1244Taxicon 25, -1248Taxicon 25, , 1987 . -The myotoxin from B. asper snake venom inhibited Ca-ATPase activity of rabbit sareoplasmie retieulum after incubation in vitro . Inhibition was non-competitive and albumin enhanced the effect of the toxin . Furthermore, B. riper myotoxin hydrolyzed sarcoplasmic reticulum phospholipids and induced a dose-dependent release of horseradish perolddase that had been trapped in sarcoplasmic reticulum vesicles . Binding studies indicated that myotoxin does not bind to any particular protein of this membrane, suggesting that the toxin might interact with phospholipids. Inhibition of Ca-ATPase is probably a consequence of an alteration in sarcoplasmic reticulum phospholipids .
The mortality rate due to snakebite envenomation in Costa Rica was estimated from 1952 to 1993. T... more The mortality rate due to snakebite envenomation in Costa Rica was estimated from 1952 to 1993. The highest mortality was observed during the 1950s and 1960s, with the highest rate of 4.83 per 100,000 population in 1953. In contrast, a rate of 0.2 per 100,000 population per year was estimated from 1990 to 1993. The most conspicuous decline in mortality occurred after 1970. The highest mortality rates were observed in the provinces of Limón and Puntarenas, especially in regions where tropical rain forests had been transformed into agricultural fields. The lowest mortality was in the province of Guanacaste, where tropical dry forest predominates and Bothrops asper (terciopelo), the most important poisonous snake in the country, is not abundant. The majority of fatalities occurred in the age groups from 10 to 19 years old. Males were more affected than females in a ratio of 3.6:1. Before 1980 most fatal cases did not receive medical attention in hospitals, whereas after 1980 the majority of cases with fatal outcome were attended in hospitals.
of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1... more of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1993.-Twenty batches of polyvalent antivenom produced at the Instituto Clodomiro Picado were analyzed for turbidity, both before and after freezing-thawing and lyophilization . Eight batches became turbid upon freezing-thawing, and this change correlated with high levels of cholesterol, triglycerides and lipoproteins, especially ß-lipoprotein. Since normal horse serum does not become turbid after freezing-thawing, despite the fact that it has high lipoprotein levels, the possibility was raised that phenol, used as a preservative during serum fractionation, might affect lipoproteins, inducing the appearance of turbidity after freezing-thawing . This hypothesis was tested by fractionating a sample of hyperimmune serum either without phenol or using two different phenol concentrations (0.1 g/dl and 0.25 g/dl). Results showed that, although the three samples had the same cholesterol and triglyceride levels before fractionation, only the one having 0.25 g/dl phenol became turbid upon freezing-thawing, containing a diffuse lipoprotein band on electrophoresis. This finding suggests that turbidity in equine antivenoms depends on the interaction of at least three factors: (a) freezing, (b) high initial cholesterol and lipoprotein concentration in the serum, and (c) addition of phenol during fractionation of serum.
The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops a... more The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa2EDTA than when receiving booster injections of venom alone, although they showed a similar antivenom response. Moreover, antivenoms produced from plasmas of horses that received booster injections of either venom alone or venom plus CaNa2EDTA had similar neutralizing activity against lethal, hemorrhagic and coagulant effects induced by B. asper venom. The similar antibody response was corroborated by Western blotting using crude venom and by an ELISA that estimates anti-myotoxin titer. It is concluded that the chelating agent CaNa2EDTA reduces the extent of local tissue damage induced by B. asper venom, without affecting the immune response of horses against pharmacologically-relevant venom components.
A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize let... more A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize lethal and myotoxic activities of Micrurus nigrocinctus venom. Both antivenoms were adjusted to a similar neutralizing potency in experiments where venom and antivenoms were preincubated prior to injection. No significant differences were observed between IgG and F(abЈ) 2 antivenoms concerning neutralization of lethal effect in rescue experiments, i.e., when antivenom was administered intravenously after envenomation. However, F(abЈ) 2 antivenom was more effective in prolonging the time of death when subneutralizing doses were administered immediately after venom injection. Both products partially reversed the binding of M. nigrocinctus ␣-neurotoxins to acetylcholine receptor in vitro. The IgG and F(abЈ) 2 antivenoms effectively neutralized venom-induced myotoxicity when administered intravenously immediately after envenomation, although neutralization was poor if antivenom injections were delayed. Intramuscular injection of venom promoted diffusion of antivenom antibodies throughout muscle tissue, and F(abЈ) 2 diffused to a higher extent than IgG molecules. Thus, despite the observation that F(abЈ) 2 antivenom was more effective than IgG antivenom in prolonging the time of death when subneutralizing doses were administered immediately after envenomation, no major differences were observed in antivenom neutralization of lethal and myotoxic effects or in their capacity to reverse neurotoxin binding to the acetylcholine receptor.
A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutral... more A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutralize lethal, hemorrhagic and coagulant activities of the venoms of 16 species of Central and South American snakes of the subfamily Crotalinae. Neutralization of lethality was studied by two dierent methods routinely used in the quality control of antivenoms at Instituto Butantan (IB) and Instituto Clodomiro Picado (ICP). Both antivenoms neutralized the majority of the venoms studied, but the values of eective doses 50% (ED 50 ) diered markedly depending on the method used. In general, higher potencies were obtained with the method of ICP, where a challenge dose corresponding to 4 LD 50 s is used, than with the method of IB, where a challenge dose of 5 LD 50 s is employed. All venoms induced hemorrhagic activity in the mouse skin test, which was eectively neutralized by the two antivenoms. All venoms, except those of Porthidium nasutum and Bothriechis lateralis, induced coagulation of human plasma in vitro and both antivenoms were eective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive
Transactions of The Royal Society of Tropical Medicine and Hygiene, 2005
A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a... more A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a mixture of the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the three most medically important snakes in sub-Saharan Africa. The antivenom is a whole IgG preparation, obtained by caprylic acid precipitation of non-IgG plasma proteins. The antivenom effectively neutralizes the most important toxic activities of the three venoms used in the immunization in standard assays involving preincubation of venom and antivenom before testing. This antivenom compares favourably with other antivenoms designed for use in Africa with respect to neutralization of the toxins present in the venom of E. ocellatus. Caprylic acid fractionation of horse hyperimmune plasma is a simple, convenient and cheap protocol for the manufacture of high quality whole IgG antivenoms. It constitutes a potentially valuable technology for the alleviation of the critical shortage of antivenom in Africa.
The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom... more The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was studied during a twelve-month period. The following parameters were evaluated: neutralizing potency against lethal activity of Bothrops asper venom; protein and phenol concentrations; pH; turbidity; safety; and sterility. Analyses were performed each month on different samples of a batch, stored at 4, 23, 30 and 37 degrees C. No significant (P greater than 0.1) variations occurred in potency, protein and phenol concentrations, pH, sterility or safety, at any of the storage temperatures during the study period. However, visual inspection revealed a moderate increase in turbidity of the samples stored at 23, 30 and 37 degrees C, at nine, four and three months, respectively. Culture of samples excluded the possibility of microbial contamination of the product leading to turbidity. Chromatographic and electrophoretic analyses demonstrated that turbidity was caused by the formation of heterogeneous protein aggregates of high molecular weight. Present results support the conclusion that, although storage temperature (up to 37 degrees C for twelve months) does not alter antivenom potency, it significantly influences the formation of protein aggregates. This phenomenon can be prevented by recommending the storage of antivenom at refrigeration temperature.
A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ec... more A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ecacy in the neutralization of several toxic and enzymatic activities of the venoms of B. lanceolatus, B. atrox and B. asper. When tested by the i.p. route in mice, B. lanceolatus venom had an LD 50 of 12.8 mg/g. In addition, it induced local tissue damage (hemorrhage, edema and myotoxicity) and showed indirect hemolytic activity, but was devoid of coagulant eect on human plasma in vitro and of de®brinating activity in mice. Antivenom was fully eective in the neutralization of lethal, hemorrhagic, edema-forming, myotoxic and indirect hemolytic eects of B. lanceolatus venom in assays involving preincubation of venom and antivenom. When tested against the venoms of B. asper and B. atrox, the antivenom completely neutralized the lethal, hemorrhagic, myotoxic and indirect hemolytic eects, and was partially eective in neutralizing edema-forming activity. In contrast, the antivenom was ineective in the neutralization of in vitro coagulant and in vivo de®brinating eects induced by these two venoms. #
Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health ha... more Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health hazard in Perú, and the intravenous administration of equine-derived antivenoms represents the only scientifically validated treatment. This study presents a preclinical assessment of the efficacy of two whole IgG antivenoms, prepared in Perú and Costa Rica, to neutralize the most relevant toxic effects induced by the venoms of Bothrops atrox, B. brazili, B. barnetti and B. pictus from Perú. Peruvian antivenom is produced by immunizing horses with Bothrops sp. venoms from this country, whereas the production of Costa Rican antivenom involves immunization with venoms from Central American snakes. The neutralization of lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating activities was evaluated in assays involving incubation of venom and antivenom prior to testing. Both antivenoms were effective in the neutralization of these effects, with quantitative variations in the values of effective dose 50% depending on the effects being studied. Peruvian antivenom was more effective in the neutralization of lethality induced by B. atrox and B. barnetti venoms. However, Peruvian antivenom failed to neutralize coagulant activity of B. barnetti venom and edema-forming activity of B. brazili venom, whereas neutralization was achieved by Costa Rican antivenom. It is concluded that an extensive immunological cross-reactivity exists between Bothrops sp. venoms from Perú and Costa Rica, and that both antivenoms are effective in the neutralization of these four venoms in a rodent model of envenoming.
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Papers by Gustavo Rojas