- Email: [email protected]
Neutralizing potency of horse antibothropic Brazilian antivenom against Bothrops snake venoms from the Amazonian rain forest
Toxicon 38 (2000) 1859±1863 www.elsevier.com/locate/toxicon
Short communication
Neutralizing potency of horse antibothropic Brazilian antivenom against Bothrops snake venoms from the Amazonian rain forest Emiro G. Muniz a, Wany S. Maria b, Maria I. EstevaÄo-Costa b, Paulo Buhrnheim a, Carlos ChaÂvez-OloÂrtegui b,* a
GereÃncia de Animais Pec° onhentos, Fundac° aÄo de Medicina Tropical, Instituto de Medicina Tropical do Amazonas, Manaus, AM, Brazil b Fundac° aÄo Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, 30510-010, Belo Horizonte, M.G., Brazil Received 20 July 1999; accepted 14 December 1999
Abstract Neutralization of lethal toxicity (50% eective dose; ED50), hemorrhagic (minimum hemorrhagic dose; MHD) and hemolytic activity (PLA2) and levels of antibodies, measured by enzyme-linked immunosorbent assay (ELISA), were investigated to test the potency of horse antibothropic serum (ABS) against Bothrops venoms from the Amazonian rain forest. ABS neutralized the lethal activity with a potency (mg of venom neutralized per 1 ml of antivenom) of 5.5, 3.7, 1.6, 1.3 and 6.5, respectively, for B. jararaca (reference venom for assessing the ABS potency in Brazil), B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. The volume of antivenom (ml) that neutralized one MHD of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms was 5, 7.71, 7.76, 8.3 and 5, respectively. ABS neutralized the PLA2 activity with a potency of 6.2, 3.2, 1.4, 2.6 and 5 respectively, for B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. ELISA reactivity of ABS against the separate venoms was found to be quite variable. The reactivity against B. jararaca venom was higher than against other Bothrops venoms. In conclusion, the assays described here suggest that Brazilian Bothrops polyspeci®c antivenom is not very ecient in neutralizing the eects of venom from some Amazonian Bothrops species. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Antivenoms; Venoms; Enzyme-linked immunosorbent assay; Neutralizing potency
* Corresponding author. Tel.: +55-31-371-9436; fax: +55-31-371-9432. E-mail address: [email protected] (C. ChaÂvez-OloÂrtegui). 0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 0 8 2 - 9
1860
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
Bothrops snake bites account for more than 90% of accidents in Brazil, thus far exceeding those produced by snakes of the genera Crotalus, Micrurus and Lachesis (Brasil, 1998). Snake bite is an important medical problem in the Amazonian region and 91.2% of snakebite patients receiving treatment at the Hospital Nelson Antunes at the Tropical Medicine Institute of Amazonas (IMT±AM) in Manaus, between August 1985 and February 1999, were bitten by snakes of the Bothrops genus (IMT±AM data bank). The majority of the snakes involved were B. atrox and 1.2% were B. brazili (Souza and Buhrnheim, 1999). B. brazili, B. bilineatus and B. taeniatus (nomenclature according to WuÈster et al., 1997) bites are more common in west Amazonia and in places south of the Amazon river (Souza and Buhrnheim, 1999). Antibothropic serum (ABS) is the speci®c treatment for envenoming by Bothrops snakes. This therapeutic antivenom is produced by the immunization of horses with venoms from a limited number of Bothrops species, with the aim of neutralizing all the Bothrops venoms. The neutralizing potency of the ABS has been traditionally assessed by studying neutralization of the lethal activity of B. jararaca venom which is considered to be the reference venom (Maria et al., 1998). The aim of the present study was to determine the neutralizing ecacy of ABS against the lethal, hemorrhagic and hemolytic activities of Bothrops venoms in the Amazonian rain forest. An indirect enzyme linked immunosorbent assay (ELISA) was also employed to compare the antigenic cross-reactivity of the ABS against these venoms. Venoms were obtained from seven B. atrox adult snakes (pool) of both sexes and measuring 100 cm in mean length, which were rescued during the inundation caused by the closure of the Balbina Dam, 100 km from Manaus, Amazonas, Brazil; also from one 123 cm long B. brazili female, one 72 cm B. bilineatus smaragdinus female and one 86 cm B. taeniatus female, collected near the high Urucu river, Coari, 500 km from Manaus. The snakes were maintained in the climatized herpetarium of the GereÃncia de Animais Pec° onhentos of the IMT±AM, Manaus, Brazil. B. jararaca venom was provided by the herpetarium of Fundac° aÄo Ezequiel Dias (FUNED), Belo Horizonte, Brazil. To collect the venom, the snakes were anaesthetized in special plastic or glass cages maintained at 28C in a CO2 atmosphere produced by dry ice evaporation. The venoms of each species were obtained by manual compression of the venom glands. These were pooled, centrifuged, ®ltered, lyophilized and stored at ÿ208C before use. Hyperimmune horse anti-Bothrops venom plasmas were obtained by conventional immunization schedules used at the DivisaÄo de ImunobioloÂgicos of FUNED. The antigen used to produce the anti-Bothrops venom plasmas consisted of venoms from B. alternatus (12.5%), B. jararaca (50%), B. jararacussu (12.5%), B. neuwiedii (12.5%) and B. moojeni (12.5%). The ABS commercially produced by FUNED (batches 940928-32 and 981021-10) was used for the ELISA and neutralization experiments. ELISA (Engvall and Perlmann,1971) was performed by coating the plates (Hemobag Produtos CiruÂrgicos Ltda, RiberaÄo Preto, SaÄo Paulo, Brazil) overnight
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
1861
at 58C with 100 ml of a 1 mg/ml preparation of crude venoms in 50 mM carbonate buer, pH 9.6. The assay was conducted as described previously (ChaÂvezOloÂrtegui et al., 1991). All measurements were made in duplicate and the results are expressed as the median of three assays. To determine the 50% median lethal dose (LD50) of the Bothrops venoms, groups of six Swiss mice were injected via the intraperitoneal (i.p.) route with dierent doses of venoms dissolved in phosphate-buered saline. Deaths were recorded up to 48 h and the LD50 was calculated by the probit analysis (Finney, 1971) using a computer program. The LD50 of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms used throughout this study were 45.5, 99, 156, 117 and 28.5 mg per 20 g mouse weight, respectively. The protective ability of the ABS was tested by separately incubating a ®xed amount (4LD50) of Bothrops venoms with various volumes of ABS for 30 min at 378C. The mixtures (0.5 ml) containing 4LD50 of the venoms were injected i.p. into groups of six mice. Death was scored at 48 h and the ED50 (50% eective dose) calculated by probit analysis as above. The protective potency was reported as the amount of venom (mg) neutralized in each ml of ABS applying the formula: P = [(4LD50ÿLD50)/ ED50] LD50 (European Pharmacopea, 1982). Hemorrhagic activity was quantitatively determined as described by Kondo et al. (1960). The lowest venom concentration which induced an hemorrhagic reaction of 10 mm in diameter 2 h after i.d. injection was de®ned as the minimum hemorrhagic dose (MHD). Neutralization of hemorrhagic activities by ABS was estimated in mice as described by SaÂnchez et al. (1991) and de®ned as the volume of serum that neutralized one MHD of each venom. PLA2 activity using an indirect hemolytic assay and inhibition of PLA2 activity by ABS was measured as described by GutieÂrrez et al. (1988). One hemolytic dose (HD) was de®ned as the lowest venom concentration (mg) which induced a hemolytic halo of 17 mm in diameter. Inhibition of PLA2 activity is expressed as ED50, de®ned as the amount of venom (mg) per 1 ml antivenom needed to reduce the PLA2 activity by 50%. The antigenic cross-reactivity, determined by indirect ELISA, of the ABS against crude B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snake venoms is shown in Fig. 1. The ABS displayed consistent immunoreactivity with the Bothrops venom antigens coated on the microtitration plate. However, the ELISA reactivity of ABS against the separate venoms was found to be quite variable. The reactivity against B. jararaca venom was higher than the reactivity against other Bothrops venoms. The potency of ABS in protecting against lethality in mice, and its ability to inhibit PLA2 and hemorrhagic activity of the B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snakes are shown in Table 1. The ABS neutralized the lethal, hemorrhagic and PLA2 activities of the venoms, albeit with dierent potencies. ABS neutralized the lethal activity with a potency of 5.5, 3.7, 1.6, 1.3 and 6.5 respectively, for B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. ABS was not very ecient in neutralizing B. atrox, B. brazili and B. bilineatus smaragdinus venoms. The minimum potency
1862
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
Fig. 1. Reactivity of Brazilian Bothrops antivenom against B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snake venoms assessed by indirect ELISA. ELISA plates were coated with 100 ml of a 1 mg/ml preparation of crude venoms from B. jararaca (Q), B. atrox (*), B. bilineatus (R), B. brazili (r) and B. taeniatus (q). Serial dilutions (1:5.000±1:160.000) of antibothropic serum were assayed for reactivity. Binding was visualized by incubation with peroxidase-conjugated anti-horse IgG (diluted 1:1000) and by the further addition of 0-phenylene diamine. Absorbance was read on a Titertek multiscan plate reader. The values given are the means of duplicates.
of ABS recommended for therapeutic use in Brazil (Brasil, 1996) is 1 ml neutralizing 5 mg of whole B. jararaca venom. The MHD of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms was 0.43, 0.74, 0.7, 1.3 and 0.33 mg per 20 g mouse weight, respectively, and the PLA2 activity for these venoms was 35, 15, 5, 15 and 55 mg, respectively. The volume of antivenom that neutralized one MHD of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms was 5, 7.71, 7.76, 8.3 and 5, respectively. Table 1 Neutralizing activity of Brazilian Bothrops antivenom against B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snake venoms Venom B. B. B. B. B. a
jararacaa atrox bilineatus brazili taeniatus
Lethality (ED50) mg/mlb
MHD (ml)c
PLA2 activity (ED50) mg/mld
5.5 3.7 1.3 1.6 6.5
5.0 7.71 7.76 8.3 5
6.2 3.2 2.6 1.4 5
B. jararaca venom is the reference venom for assessing the bothropic antivenom potency in Brazil. Amount of venom (mg) per 1 ml antivenom needed to prevent death in 50% of injected mice. c Volume of antivenom that neutralizes one MHD (minimum hemorrhagic dose) of each venom. d Amount of venom (mg) per 1 ml antivenom needed to reduce by 50% the PLA2 activity of crude venom. b
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
1863
ABS neutralized the PLA2 activity with a potency (mg of venom neutralized by 1 ml of antivenom) of 6.2, 3.2, 1.4, 2.6 and 5, respectively, for B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms.The ABS was also more ecient in neutralizing the PLA2 activity from B. jararaca venom. In conclusion, the mouse protection test, combined with the other in vitro assays described here, suggests that Brazilian Bothrops polyspeci®c antivenom is not very ecient in neutralizing the eects of venoms of some Bothrops species from the Amazonian rain forest. The ecacy of another antivenom used as antigen for producing horse anti-Bothrops venom plasmas from B. atrox, B. bilineatus, B. brazili and B. taeniatus venoms is currently under investigation. Acknowledgements This research was supported by grants from Conselho Nacional de Desenvolvimento CientõÂ ®co e TecnoloÂgico (CNPq) and Fundac° aÄo de Amparo aÁ Pesquisa do Estado de Minas Gerais (FAPEMIG). C.C.O. is the recipient of a CNPq fellowship. References Brasil, DiaÂrio O®cial da UniaÄo 1996. Portaria No. 174 de 11 Nov. Normas de produc° aÄo e controle de qualidade dos soros antiofõÂ dicos, antitoÂxicos e antiraÂbico. Brasilia, Imprensa Nacional. Brasil, MinisteÂrio da SauÂde, FNS 1998. Manual de DiagnoÂstico e Tratamento de Acidentes por Animais Pec° onhentos. 131n pp, BrasõÂ lia, FNS. Chavez-OloÂrtegui, C., Ait Amara, D., Rochat, H., Diniz, C., Granier, C., 1991. In vivo protection against scorpion toxins by liposomal immunization. Vaccine 9, 907±910. Engvall, E., Perlmann, P., 1971. Enzyme linked immunosorbent assay (ELISA): quantitative assay of immunoglobulin G. Immunochem. 8, 871±874. Sainte, Rune, Maisonneuv, (Eds), 1982, European Pharmacopea, 145.1±145.3. Finney, D.J., 1971. Probit Analysis. Cambridge University, Cambridge. GutieÂrrez, J.M., Avila, C., Rojas, E., Cerdas, L., 1988. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411±413. Maria, W.S., Cambuy, M.O., Costa, J.O., Velarde, D.T., ChaÂvez-OloÂrtegui, C., 1998. Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods. Toxicon 36 (10), 1433±1439. Kondo, H., Kondo, S.I., Kegawa, H., Murata, R., Oshaka, A., 1960. Studies on the quantitative method for determination of hemorrhagic activity of Habu snake venom. Jph. J. Med. Sci. Biol. 13, 43±51. SaÂnchez, E.F., MagalhaÄes, A., Mandelbaum, F.R., Diniz, C.R., 1991. Puri®cation and characterization of the hemorrhagic factor from the venom of the bushmaster snake (Lachesis muta muta ). Biochim. Biophys. Acta 1074, 347±354. Souza, A.R.B., Buhrnheim, P.F., 1999. TreÃs acidentes por Bothrops brazili Hoge atendidos no IMT± AM de 1986 a 1995. Revta Soc. Brasil. Med. Trop. 32 (Suppl. I), 388. WuÈster, W., Golay, P., Warrel, D.A., 1997. Synopsis of recent developments in venomous snake systematics. Toxicon 35 (3), 319±340.
Short communication
Neutralizing potency of horse antibothropic Brazilian antivenom against Bothrops snake venoms from the Amazonian rain forest Emiro G. Muniz a, Wany S. Maria b, Maria I. EstevaÄo-Costa b, Paulo Buhrnheim a, Carlos ChaÂvez-OloÂrtegui b,* a
GereÃncia de Animais Pec° onhentos, Fundac° aÄo de Medicina Tropical, Instituto de Medicina Tropical do Amazonas, Manaus, AM, Brazil b Fundac° aÄo Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, 30510-010, Belo Horizonte, M.G., Brazil Received 20 July 1999; accepted 14 December 1999
Abstract Neutralization of lethal toxicity (50% eective dose; ED50), hemorrhagic (minimum hemorrhagic dose; MHD) and hemolytic activity (PLA2) and levels of antibodies, measured by enzyme-linked immunosorbent assay (ELISA), were investigated to test the potency of horse antibothropic serum (ABS) against Bothrops venoms from the Amazonian rain forest. ABS neutralized the lethal activity with a potency (mg of venom neutralized per 1 ml of antivenom) of 5.5, 3.7, 1.6, 1.3 and 6.5, respectively, for B. jararaca (reference venom for assessing the ABS potency in Brazil), B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. The volume of antivenom (ml) that neutralized one MHD of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms was 5, 7.71, 7.76, 8.3 and 5, respectively. ABS neutralized the PLA2 activity with a potency of 6.2, 3.2, 1.4, 2.6 and 5 respectively, for B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. ELISA reactivity of ABS against the separate venoms was found to be quite variable. The reactivity against B. jararaca venom was higher than against other Bothrops venoms. In conclusion, the assays described here suggest that Brazilian Bothrops polyspeci®c antivenom is not very ecient in neutralizing the eects of venom from some Amazonian Bothrops species. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Antivenoms; Venoms; Enzyme-linked immunosorbent assay; Neutralizing potency
* Corresponding author. Tel.: +55-31-371-9436; fax: +55-31-371-9432. E-mail address: [email protected] (C. ChaÂvez-OloÂrtegui). 0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 0 8 2 - 9
1860
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
Bothrops snake bites account for more than 90% of accidents in Brazil, thus far exceeding those produced by snakes of the genera Crotalus, Micrurus and Lachesis (Brasil, 1998). Snake bite is an important medical problem in the Amazonian region and 91.2% of snakebite patients receiving treatment at the Hospital Nelson Antunes at the Tropical Medicine Institute of Amazonas (IMT±AM) in Manaus, between August 1985 and February 1999, were bitten by snakes of the Bothrops genus (IMT±AM data bank). The majority of the snakes involved were B. atrox and 1.2% were B. brazili (Souza and Buhrnheim, 1999). B. brazili, B. bilineatus and B. taeniatus (nomenclature according to WuÈster et al., 1997) bites are more common in west Amazonia and in places south of the Amazon river (Souza and Buhrnheim, 1999). Antibothropic serum (ABS) is the speci®c treatment for envenoming by Bothrops snakes. This therapeutic antivenom is produced by the immunization of horses with venoms from a limited number of Bothrops species, with the aim of neutralizing all the Bothrops venoms. The neutralizing potency of the ABS has been traditionally assessed by studying neutralization of the lethal activity of B. jararaca venom which is considered to be the reference venom (Maria et al., 1998). The aim of the present study was to determine the neutralizing ecacy of ABS against the lethal, hemorrhagic and hemolytic activities of Bothrops venoms in the Amazonian rain forest. An indirect enzyme linked immunosorbent assay (ELISA) was also employed to compare the antigenic cross-reactivity of the ABS against these venoms. Venoms were obtained from seven B. atrox adult snakes (pool) of both sexes and measuring 100 cm in mean length, which were rescued during the inundation caused by the closure of the Balbina Dam, 100 km from Manaus, Amazonas, Brazil; also from one 123 cm long B. brazili female, one 72 cm B. bilineatus smaragdinus female and one 86 cm B. taeniatus female, collected near the high Urucu river, Coari, 500 km from Manaus. The snakes were maintained in the climatized herpetarium of the GereÃncia de Animais Pec° onhentos of the IMT±AM, Manaus, Brazil. B. jararaca venom was provided by the herpetarium of Fundac° aÄo Ezequiel Dias (FUNED), Belo Horizonte, Brazil. To collect the venom, the snakes were anaesthetized in special plastic or glass cages maintained at 28C in a CO2 atmosphere produced by dry ice evaporation. The venoms of each species were obtained by manual compression of the venom glands. These were pooled, centrifuged, ®ltered, lyophilized and stored at ÿ208C before use. Hyperimmune horse anti-Bothrops venom plasmas were obtained by conventional immunization schedules used at the DivisaÄo de ImunobioloÂgicos of FUNED. The antigen used to produce the anti-Bothrops venom plasmas consisted of venoms from B. alternatus (12.5%), B. jararaca (50%), B. jararacussu (12.5%), B. neuwiedii (12.5%) and B. moojeni (12.5%). The ABS commercially produced by FUNED (batches 940928-32 and 981021-10) was used for the ELISA and neutralization experiments. ELISA (Engvall and Perlmann,1971) was performed by coating the plates (Hemobag Produtos CiruÂrgicos Ltda, RiberaÄo Preto, SaÄo Paulo, Brazil) overnight
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
1861
at 58C with 100 ml of a 1 mg/ml preparation of crude venoms in 50 mM carbonate buer, pH 9.6. The assay was conducted as described previously (ChaÂvezOloÂrtegui et al., 1991). All measurements were made in duplicate and the results are expressed as the median of three assays. To determine the 50% median lethal dose (LD50) of the Bothrops venoms, groups of six Swiss mice were injected via the intraperitoneal (i.p.) route with dierent doses of venoms dissolved in phosphate-buered saline. Deaths were recorded up to 48 h and the LD50 was calculated by the probit analysis (Finney, 1971) using a computer program. The LD50 of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms used throughout this study were 45.5, 99, 156, 117 and 28.5 mg per 20 g mouse weight, respectively. The protective ability of the ABS was tested by separately incubating a ®xed amount (4LD50) of Bothrops venoms with various volumes of ABS for 30 min at 378C. The mixtures (0.5 ml) containing 4LD50 of the venoms were injected i.p. into groups of six mice. Death was scored at 48 h and the ED50 (50% eective dose) calculated by probit analysis as above. The protective potency was reported as the amount of venom (mg) neutralized in each ml of ABS applying the formula: P = [(4LD50ÿLD50)/ ED50] LD50 (European Pharmacopea, 1982). Hemorrhagic activity was quantitatively determined as described by Kondo et al. (1960). The lowest venom concentration which induced an hemorrhagic reaction of 10 mm in diameter 2 h after i.d. injection was de®ned as the minimum hemorrhagic dose (MHD). Neutralization of hemorrhagic activities by ABS was estimated in mice as described by SaÂnchez et al. (1991) and de®ned as the volume of serum that neutralized one MHD of each venom. PLA2 activity using an indirect hemolytic assay and inhibition of PLA2 activity by ABS was measured as described by GutieÂrrez et al. (1988). One hemolytic dose (HD) was de®ned as the lowest venom concentration (mg) which induced a hemolytic halo of 17 mm in diameter. Inhibition of PLA2 activity is expressed as ED50, de®ned as the amount of venom (mg) per 1 ml antivenom needed to reduce the PLA2 activity by 50%. The antigenic cross-reactivity, determined by indirect ELISA, of the ABS against crude B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snake venoms is shown in Fig. 1. The ABS displayed consistent immunoreactivity with the Bothrops venom antigens coated on the microtitration plate. However, the ELISA reactivity of ABS against the separate venoms was found to be quite variable. The reactivity against B. jararaca venom was higher than the reactivity against other Bothrops venoms. The potency of ABS in protecting against lethality in mice, and its ability to inhibit PLA2 and hemorrhagic activity of the B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snakes are shown in Table 1. The ABS neutralized the lethal, hemorrhagic and PLA2 activities of the venoms, albeit with dierent potencies. ABS neutralized the lethal activity with a potency of 5.5, 3.7, 1.6, 1.3 and 6.5 respectively, for B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. ABS was not very ecient in neutralizing B. atrox, B. brazili and B. bilineatus smaragdinus venoms. The minimum potency
1862
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
Fig. 1. Reactivity of Brazilian Bothrops antivenom against B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snake venoms assessed by indirect ELISA. ELISA plates were coated with 100 ml of a 1 mg/ml preparation of crude venoms from B. jararaca (Q), B. atrox (*), B. bilineatus (R), B. brazili (r) and B. taeniatus (q). Serial dilutions (1:5.000±1:160.000) of antibothropic serum were assayed for reactivity. Binding was visualized by incubation with peroxidase-conjugated anti-horse IgG (diluted 1:1000) and by the further addition of 0-phenylene diamine. Absorbance was read on a Titertek multiscan plate reader. The values given are the means of duplicates.
of ABS recommended for therapeutic use in Brazil (Brasil, 1996) is 1 ml neutralizing 5 mg of whole B. jararaca venom. The MHD of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms was 0.43, 0.74, 0.7, 1.3 and 0.33 mg per 20 g mouse weight, respectively, and the PLA2 activity for these venoms was 35, 15, 5, 15 and 55 mg, respectively. The volume of antivenom that neutralized one MHD of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms was 5, 7.71, 7.76, 8.3 and 5, respectively. Table 1 Neutralizing activity of Brazilian Bothrops antivenom against B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus snake venoms Venom B. B. B. B. B. a
jararacaa atrox bilineatus brazili taeniatus
Lethality (ED50) mg/mlb
MHD (ml)c
PLA2 activity (ED50) mg/mld
5.5 3.7 1.3 1.6 6.5
5.0 7.71 7.76 8.3 5
6.2 3.2 2.6 1.4 5
B. jararaca venom is the reference venom for assessing the bothropic antivenom potency in Brazil. Amount of venom (mg) per 1 ml antivenom needed to prevent death in 50% of injected mice. c Volume of antivenom that neutralizes one MHD (minimum hemorrhagic dose) of each venom. d Amount of venom (mg) per 1 ml antivenom needed to reduce by 50% the PLA2 activity of crude venom. b
E.G. Muniz et al. / Toxicon 38 (2000) 1859±1863
1863
ABS neutralized the PLA2 activity with a potency (mg of venom neutralized by 1 ml of antivenom) of 6.2, 3.2, 1.4, 2.6 and 5, respectively, for B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms.The ABS was also more ecient in neutralizing the PLA2 activity from B. jararaca venom. In conclusion, the mouse protection test, combined with the other in vitro assays described here, suggests that Brazilian Bothrops polyspeci®c antivenom is not very ecient in neutralizing the eects of venoms of some Bothrops species from the Amazonian rain forest. The ecacy of another antivenom used as antigen for producing horse anti-Bothrops venom plasmas from B. atrox, B. bilineatus, B. brazili and B. taeniatus venoms is currently under investigation. Acknowledgements This research was supported by grants from Conselho Nacional de Desenvolvimento CientõÂ ®co e TecnoloÂgico (CNPq) and Fundac° aÄo de Amparo aÁ Pesquisa do Estado de Minas Gerais (FAPEMIG). C.C.O. is the recipient of a CNPq fellowship. References Brasil, DiaÂrio O®cial da UniaÄo 1996. Portaria No. 174 de 11 Nov. Normas de produc° aÄo e controle de qualidade dos soros antiofõÂ dicos, antitoÂxicos e antiraÂbico. Brasilia, Imprensa Nacional. Brasil, MinisteÂrio da SauÂde, FNS 1998. Manual de DiagnoÂstico e Tratamento de Acidentes por Animais Pec° onhentos. 131n pp, BrasõÂ lia, FNS. Chavez-OloÂrtegui, C., Ait Amara, D., Rochat, H., Diniz, C., Granier, C., 1991. In vivo protection against scorpion toxins by liposomal immunization. Vaccine 9, 907±910. Engvall, E., Perlmann, P., 1971. Enzyme linked immunosorbent assay (ELISA): quantitative assay of immunoglobulin G. Immunochem. 8, 871±874. Sainte, Rune, Maisonneuv, (Eds), 1982, European Pharmacopea, 145.1±145.3. Finney, D.J., 1971. Probit Analysis. Cambridge University, Cambridge. GutieÂrrez, J.M., Avila, C., Rojas, E., Cerdas, L., 1988. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411±413. Maria, W.S., Cambuy, M.O., Costa, J.O., Velarde, D.T., ChaÂvez-OloÂrtegui, C., 1998. Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods. Toxicon 36 (10), 1433±1439. Kondo, H., Kondo, S.I., Kegawa, H., Murata, R., Oshaka, A., 1960. Studies on the quantitative method for determination of hemorrhagic activity of Habu snake venom. Jph. J. Med. Sci. Biol. 13, 43±51. SaÂnchez, E.F., MagalhaÄes, A., Mandelbaum, F.R., Diniz, C.R., 1991. Puri®cation and characterization of the hemorrhagic factor from the venom of the bushmaster snake (Lachesis muta muta ). Biochim. Biophys. Acta 1074, 347±354. Souza, A.R.B., Buhrnheim, P.F., 1999. TreÃs acidentes por Bothrops brazili Hoge atendidos no IMT± AM de 1986 a 1995. Revta Soc. Brasil. Med. Trop. 32 (Suppl. I), 388. WuÈster, W., Golay, P., Warrel, D.A., 1997. Synopsis of recent developments in venomous snake systematics. Toxicon 35 (3), 319±340.