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Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica
Toxicon 38 (2000) 633±644 www.elsevier.com/locate/toxicon
Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica I. Fernandes a,*, E.X. Lima a, H.A. Takehara a, A.M. Moura-da-Silva a, I. Tanjoni a, J.M. GutieÂrrez b a
LaboratoÂrio de Imunopatologia, Instituto Butantan, SaÄo Paulo, Brazil Instituto Clodomiro Picado, Facultad de MicrobiologõÂa, Universidad de Costa Rica, San JoseÂ, Costa Rica
b
Received 18 May 1999; accepted 6 July 1999
Abstract Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000±000. This work compared the speci®city, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys ) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus ) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by anity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG pro®le and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic
* Corresponding author at: LaboratoÂrio de Imunopatologia, Instituto Butantan, Av. Vital Brazil 1500, 05503-900, SaÄo Paulo, Brazil. Fax: +55-11-815-1505. E-mail address: [email protected] (I. Fernandes). 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 7 7 - 4
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antivenom of Instituto Butantan being more eective. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: IgG isotypes; Cross-neutralization; Snake antivenoms; Horse
1. Introduction Snakes of the genus Bothrops are widespread in Latin America, and are responsible for most snakebites in this region (MinisteÂrio da SauÂde, 1990; Fan and Cardoso, 1995; GutieÂrrez, 1995). In Brazil, species of Bothrops cause more than 70% of accidents by snakebites (MinisteÂrio da SauÂde, 1990) and Bothrops jararaca is involved in many of them. On the other hand, B. asper is the most important species in Central America from a medical point of view (BolanÄos, 1982, GutieÂrrez, 1995). Bothrops sp envenomations are characterized by in¯ammation, edema and extensive local tissue damage at the site of the bite, together with systemic eects such as hemorrhage, coagulopathies, renal failure and cardiovascular shock (Rosenfeld, 1971; GutieÂrrez and Lomonte, 1989; Fan and Cardoso, 1995). Antivenoms obtained from hyperimmunized horse plasmas constitute the only speci®c treatment for snakebite envenomation. Despite long-term experience with antivenom therapy, relatively little is known about horse antivenom IgG isotypes. It is of relevance to know the IgG isotype pro®le of antivenoms as well as the role of each isotype in toxin neutralization, since ideally only the eective isotypes should be administered to patients. Horse IgG isotypes have been designated IgGa, IgGb, IgGc and IgG(T), based on their immunoelectrophoretic mobility (Klinman et al., 1965). In order to clarify the role of major isotypes present in bothropic and crotalic antivenoms from Instituto Butantan, IgG(T) and IgGa have been isolated and their neutralizing ability evaluated (Fernandes et al., 1991, 1994 and 1997). These two isotypes contained most of the antivenom speci®c antibodies responsible for the neutralization of lethality and other important venom activities. Antivenoms are currently produced in many laboratories around the world (Theakston and Warrell, 1991), with very dierent immunization protocols and methodologies for immunoglobulin puri®cation. Two of the major producers in Latin America are the Instituto Butantan in Brazil and Instituto Clodomiro Picado in Costa Rica. The former produces a bothropic antivenom by immunizing horses with venoms of ®ve species of Bothrops sp predominant in the southeastern Brazil (Raw et al., 1991) whereas Instituto Clodomiro Picado produces a polyvalent antivenom with an immunization mixture containing venoms of Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys (BolanÄos and Cerdas, 1980). It is therefore important to study if the dierences in the type of venoms used for immunization, as well as in the protocols followed, result in antivenoms with dierent neutralizing capacities and IgG isotype pro®le.
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The present study compared antibody titers, speci®city and IgG subclass content of the polyvalent antivenom of Instituto Clodomiro Picado and the bothropic antivenom of Instituto Butantan which are used in these countries in the treatment of Bothrops sp envenomations. In addition, the major isotypes IgG(T) and IgGa were isolated from both antivenoms and a comparative analysis was conducted on their neutralizing ecacy against lethal, coagulant, hemorrhagic and phospholipase A2 activities of the venoms. Finally, the cross neutralization of both antivenoms against B. jararaca and B. asper venoms was also evaluated. 2. Materials and methods 2.1. Animals Two-month-old male Swiss mice, weighing 18±22 g, were used. 2.2. Venoms B. jararaca venom was extracted from snakes held at the Instituto Butantan and B. asper venom from specimens kept at Instituto Clodomiro Picado, Costa Rica. Venoms were lyophilized and stored at ÿ208C. 2.3. Antivenoms The polyvalent antivenom of Instituto Clodomiro Picado (ICP) (anti-B. asper, C. d. durissus and L. m. stenophrys ) and the bothropic antivenom of Instituto Butantan (IB) (anti-B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus ) were used throughout. Immunization protocols and adjuvants used have been previously described (Raw et al., 1991; Angulo et al., 1997). Goat antihorse IgG(T) was purchased from ICN Immunobiologicals (Lisle, IL, U.S.A.) and rabbit anti-horse IgGa, IgGb, IgGc were purchased from Bethyl Laboratories INC (Montgomery, ALA, U.S.A.). 2.4. Isolation of horse IgG(T) and IgGa from antivenom IgGa and IgG(T) isotypes were isolated from IB or ICP antivenoms by chromatography on protein A-Sepharose followed by chromatography on a column of anti-horse IgG(T) monoclonal antibody (LO-HoGT-1)-Sepharose as reported by Fernandes et al. (1994). Brie¯y, a 2.5 cm 8.0 cm protein ASepharose column was equilibrated with 0.15 M borate buered saline, pH 8.0 (BBS) and 0.5 ml of ICP or IB antivenom was applied to the column which was washed with BBS until no protein could be detected in the euent. The bound IgG isotypes were then eluted with 0.15 M citrate buer pH 5.5. All procedures were performed at 48C. Immunochemical analysis of the material eluted at pH 5.5 using rabbit anti-horse immunoglobulins showed that it contained only IgG(T)
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and IgGa. Separation of the isotypes was then achieved by passing the eluted mixture in the column of Sepharose-LO-HoGT-1. The ®rst peak obtained by ¯ushing the column with BBS contained IgGa whereas the second peak, eluted with 0.15 M citrate buer pH 5.0, contained IgG(T). The purity of these fractions was evaluated by immunoelectrophoresis. The IgG(T) and IgGa isotypes isolated from horse IB or ICP antivenom were then used to ®nd out their ability to neutralize lethality of B. jararaca or B. asper venoms, respectively. 2.5. Antibody assay (ELISA) The antibody titers of the dierent IgG subclasses of IB and ICP antivenoms to B. jararaca or B. asper venoms were determined by ELISA according to the method of Theakston et al. (1977), with some modi®cations. Brie¯y, 50 mg/ml venom were used to coat the plates and IB or ICP antivenom dilutions were added followed by 1 mg/ml goat anti-horse IgGa, IgGb, IgGc or IgG(T). Next, peroxidase labeled anti-goat Ig6 (Sigma) was added followed by orthophenylene diamine plus H2O2 as substrate. Absorbances were recorded in an ELISA reader (Multiskan spectrophotometer, EFLAB, Helsinski, Finland) and the titers were determined as the reciprocal of the highest dilution that causes an absorbance greater than 0.100 at 492 nm, as non-speci®c reactions were observed below this value. 2.6. Determination of LD50 The i.p. LD50 for B. jararaca (33 mg/20 g mouse) and for B. asper (77.5 mg/20 g mouse) venoms were determined by probit test (Finney, 1971) using groups of six mice at each dose. Survival was recorded 48 h after venom injection. The data were analyzed according to Almeida et al. (1990). 2.7. Neutralization of lethality Neutralizing ability of ICP or IB antivenom is expressed as eective dose 50% (ED50), de®ned as microlitres of antivenom per milligram of venom at which half of the injected animals survived (GutieÂrrez et al., 1990). The challenge dose of venom used was three LD50. The ED50 for IgGa and IgG(T) is de®ned as milligram of protein per milligram of venom. Samples were mixed with venom dissolved in saline. The mixtures were incubated for 30 min at 378C, centrifuged to remove the immune complexes and the supernatant injected i.p. into mice. Control animals received the same amount of venom dissolved in the same volume of saline without antibodies. Survival was determined 48 h later. 2.8. Antihemorrhagic activity The method of Kondo et al. (1960), as modi®ed by GutieÂrrez et al. (1985), was used. Brie¯y, a constant amount of venom was incubated with various dilutions of
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antibodies, in order to obtain several antibody/venom ratios. Mixtures were incubated for 30 min at 378C and aliquots of 0.1 ml containing 10 minimum hemorrhagic doses (MHD, amount of venom that induces a hemorrhagic lesion of 10 mm diameter 2 h after injection) were injected i.d. in the abdominal region of groups of four Swiss mice. Control mice were injected with either venom alone or antibodies alone. Two hours after injection animals were sacri®ced, their skin removed and the hemorrhagic areas in the inner side of the skin measured. Neutralization was expressed as ED50, de®ned as the ratio mg antibody/mg venom in which the eect of venom alone was neutralized by 50%. 2.9. Neutralization of coagulant activity The method of Theakston and Reid (1983), as modi®ed by Gene et al. (1989) was followed, using human citrated plasma. A constant amount of venom was incubated with various dilutions of antibodies. Mixtures were incubated as described above. Then, 0.1 ml of the mixtures, containing two minimum coagulant doses of venom (MCD, minimal amount of venom that induces coagulation of plasma in 60 s) were added to samples of 0.2 ml of citrated plasma and the clotting times recorded. In control tubes plasma was incubated with either venom alone or antibodies alone. Neutralization was expressed as eective dose (ED), de®ned as the ratio mg antibodies/mg venom at which the clotting time increased three times when compared with clotting time of plasma incubated with two MCD of venom alone (Gene et al., 1989). 2.10. Neutralization of PLA2 activity (indirect hemolysis) The method described by GutieÂrrez et al. (1988) was used. Brie¯y, a constant amount of venom was incubated with various dilutions of antibodies and mixtures were incubated as described before. Then, aliquots of 10 ml of the mixtures were added to wells in agarose-egg yolk-sheep erythrocyte gels (GutieÂrrez et al., 1988). Control samples included venom incubated without antibodies and antibodies incubated without venom. Plates were incubated at 378C for 20 h and neutralization expressed as the ratio mg antibodies/mg venom able to reduce by 50% the diameter of the hemolytic halo when compared to the eect induced by venom alone. 2.11. Western blotting Venom antigens were separated by electrophoresis according to the method of Laemmli (1970) using 8 to 18% polyacrylamide gel plus SDS (SDS-PAGE) under non-reducing conditions. The gels were either stained with 0.2% (w/v) Coomassie Brilliant Blue G-250 in methanol:water:acetic acid (4:5:1) or transferred at 100 V for 2 h to a 0.45 mm pore size nitrocellulose membrane using the Pharmacia (Uppsala, Sweden) Trans-Blot system by the procedure of Towbin et al. (1979). The nitrocellulose membrane was cut into strips and incubated for 2 h in PBS
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plus 5% defatted milk to block unoccupied sites. All incubations and washing steps were performed on an end-to-end rotor at room temperature. The strips were incubated with a 1:5,000 dilution of IB or ICP antivenom for 1 h. Some strips were incubated with conjugated rabbit anti-horse IgG peroxidase and developed while others were incubated with goat anti-horse IgGa, IgGb, IgGc or IgG(T) followed by rabbit anti-goat IgG and then developed by the addition of freshly prepared 4a-chloro-1-a-naphtol at 3 mg/ml plus hydrogen peroxide. The reaction was allowed to proceed for 1±5 min and stopped by washing strips with water. Lysozyme 14,300, b-lactoglobulin 18,300, carbonic anhydrase 29,000, ovalbumin 46,900, bovine serum albumin 68,000, phosphorylase B 97,400 and myosin-H 200,000 were used as mol. wt markers. 3. Results IB and ICP antivenoms are produced under dierent immunization schedules using as antigens the predominant Bothrops species in Brazil and Costa Rica, respectively. First, we compared the antibody titer, speci®city and neutralizing ability of both antivenoms when confronted with either the homologous venom (used in the immunization protocol of the antivenom in test) or the heterologous one (present in the immunization protocol of the other antivenom). Despite the dierences in the immunization protocol, both antivenoms presented similar antibody titers against the homologous venom when assayed by ELISA, the same being observed for their IgG isotypes (Table 1). Interestingly, high antibody titers were also detected when whole antivenoms were assayed using heterologous venoms. In these cases, the dierences in antibody titers were around two dilution steps (Table 1). The IgG isotypes were also similar for both IB and ICP antivenoms: IgG(T) is the major isotype for antivenom antibodies, followed by IgGa, with smaller titers for IgGb and IgGc (Table 1). The speci®city of ICP and IB antivenoms was also compared. B. asper and B. jararaca venoms dier considerably in their electrophorectic pattern with the Table 1 Antibody titers of IB and ICP antivenoms and their dierent IgG subclasses to B. jararaca and B. asper venomsa
Whole antivenom IgG(T) IgGa IgGb IgGc
IB (B. jararaca )
IB (B. asper )
ICP (B. asper )
ICP (B. jararaca )
1,024,000 1,024,000 64,000 16,000 16,000
1,024,000 512,000 256,000 64,000 32,000
512,000 512,000 128,000 32,000 8,000
256,000 128,000 64,000 16,000 4,000
a The antibody titers were determined by ELISA as the reciprocal of the highest dilution with an absorbance greater than 0.100 at 492 nm, since non-speci®c reactions were observed below this value.
Fig. 1. Western blotting analysis of whole antivenoms (A) and IgG isotypes of IB (B) and ICP (C) antivenoms. Approximately 5 mg of B. asper (Ba) or B. jararaca (Bj) venoms were resolved by SDS-PAGE using 8 to 18% under non-reducing conditions and either stained by Coomassie blue (cb) or transferred to nitrocellulose membrane. After blocking unoccupied sites the membrane was incubated with IB or ICP antivenom, followed by rabbit anti-horse IgG peroxidase conjugate or goat anti-horse IgGa (a), IgGb (b), IgGc (c) or IgG(T) (T) followed by rabbit anti-goat IgG conjugate. Arrows on the left indicate the migration of mol. wt markers.
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predominance of low mol. wt. bands in the former and high mol. wt. bands in the latter (Fig. 1(A), lines Ba/cb and Bj/cb). Despite that, ICP and IB antivenoms recognized the same pattern by immunoblotting in each venom (Fig. 1(A)). The speci®city of the antibodies contained in each isotype was also consistent. There was no dierence in the ability of each IgG subclass to recognize venom antigens. However, IgG(T) gave a stronger reaction, followed decreasingly by IgGa, IgGb and IgGc, these results being in agreement with the antibody titers observed by ELISA. The same results were observed for IB (Fig. 1(B)) and ICP (Fig. 1(C)) antivenoms. The neutralizing capacity of both antivenoms for homologous and heterologous venoms was also determined. As shown in Table 2, ICP and IB antivenoms had a similar ability to neutralize homologous venoms. However, the polyvalent antivenom of ICP neutralized the heterologous B. jararaca venom less eciently than that of B. asper. Signi®cant dierences were also detected in the neutralization of B. jararaca venom by IB and ICP antivenoms, the ®rst being more ecient. Interestingly, the neutralization of B. asper venom was achieved similarly by homologous and heterologous antivenoms (Table 2). The IgGa and IgG(T) isotypes were isolated from both antivenoms by a combination of two anity chromatographic procedures, protein A-Sepharose followed by anti-IgG(T) mAb (LO-HoGT-1)-Sepharose and tested against the homologous venoms. The neutralizing ability of these subclasses is also shown in Table 2. IgG(T) was always more eective than IgGa from IB or ICP antivenoms. In IB antivenom, the relative concentration of neutralizing antibodies is at least 3 times higher in IgG(T) than in IgGa. In ICP antivenom, neutralizing antibodies were almost exclusively of IgG(T) isotype (Table 2). Table 2 Ability of IB and ICP antivenoms and their IgG(T) and IgGa subclasses to neutralize lethality in mice induced by B. jararaca and B. asper venomsa B. jararaca IB Antivenomb
B. asper
90.1 (70.5±109.6) 4.1 (3.74.4) 13.3 (9.7±18.7)
83.8 (76.6±90.5) ND
IgG(T)c
284.7 (222.2±331.3) ND
IgGac
ND
114.6 (74.8±141.6) 4.6 (4.0±5.3) > 13.0
IgG(T)c IgGab ICP Antivenomb
ND
a The neutralizing ability was expressed as the ED50, de®ned as microliters of antivenoms b or milligrams of isolated immunoglobulin isotypes c per milligram of venom. ND: not determined.
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Table 3 Neutralization of hemorrhage, coagulation and indirect hemolysis induced by B. asper and B. jararaca venoms using IgG(T) and IgGa isolated from IB and ICP antivenomsa
Hemorrhage Coagulation Indirect hemolysis a
Neutralization of B. jararaca venom by IB antivenom
Neutralization of B. asper venom by ICP antivenom
IgG(T)
IgGa
IgG(T)
IgGa
4.420.5 5.320.4 4.120.5
12.421.1 11.221.2 12.321.4
1.920.1 12.020.8 28.023.0
8.120.7 16.0 20.9 > 40.0
Neutralization of the activities is expressed as mean2SD (mg antibody/mg venom).
IgGa and IgG(T) isotypes were also tested regarding their ability to neutralize hemorrhagic, coagulant and PLA2 (indirect hemolytic) activities. The results of these experiments are shown in Table 3. In both antivenoms IgG(T) was the major isotype responsible for neutralization of these activities, followed by IgGa. 4. Discussion Although antivenoms derived from hyperimmune horse plasma have been used for over a century in the treatment of snakebite envenomation, little is known about the content of various IgG isotypes in these immunobiologicals, as well as on the neutralizing ability of the dierent isotypes. Antivenoms produced in Brazil and Costa Rica are used in several Latin American countries, and eorts are being made to gain a better knowledge and understanding of antivenom characteristics and neutralizing pro®le. Despite important dierences in the immunization protocols used at Instituto Clodomiro Picado and Instituto Butantan, as well as in the venoms used, both antivenoms presented similar antibody titers and speci®cities for the various IgG isotypes studied. Nevertheless, slight dierences were noticed, since IgGa from ICP antivenom failed to neutralize phospholipase A2 activities and lethality induced by B. asper venom. Several factors may control the levels of each isotype during the immune response. In mice and humans, the restriction of antibody isotype response is associated with activation of speci®c subpopulations of Thelper cells which present a dierent pattern of cytokine expression (Powrie and Coman, 1993). However, little is know about isotype control in horse immune response. Some components in the venoms of C. d. terri®cus and L. m. muta have been associated with alterations in the immune response in mice (Cardoso and Mota, 1997; Stephano et al., 1996). Crotoxin, the main protein of C. d. terri®cus venom is a potent immunesupressor, aecting the proliferation of lymphocytes and the cytokine balance (Cardoso et al., 1998). Immunosupression was also observed with L. m. muta venom when mice were immunized with various antigens
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(Stephano et al., 1996). Interestingly, the presence of Crotalus and Lachesis venoms in the immunizing mixture of polyvalent antivenom did not seem to alter the titer or the isotype pro®le of the product. However, the presence of these components may be related to the reduction in the neutralizing ability of IgGa. This suggestion is supported by the observation that anti-crotalic IgGa is seven times less potent than IgG(T) while anti-bothropic IgGa is only three times (Fernandes et al., 1997). Regarding the neutralizing ability of the isotypes, IgG(T) was more ecient than IgGa, on a weight per weight basis, in the neutralization of lethal, hemorrhagic, coagulant and indirect hemolytic activities. This agrees with previous ®ndings of Fernandes et al. (1997) and con®rms that IgG(T) is the most important immunoglobulin in bothropic and crotalic antivenoms. On this basis, some manufacturers have introduced chromatographic steps in their production protocols aimed at the puri®cation of IgG(T), with the consequent reduction in total protein concentration of antivenoms while keeping a high neutralizing titer (Grandgeorge et al., 1996). Our observations showed a strong cross-neutralization of IB and ICP antivenoms when confronted with heterologous venoms. Immunological crossreactivity in venom±antivenom systems has been well documented (Mebs et al., 1988; Russell, 1988; Ferreira et al., 1992; GutieÂrrez et al., 1996; de Roodt et al., 1998). In contrast, BogarõÂ n et al. (1995) described that three heterologous antivenoms produced in dierent Latin American countries failed to neutralize lethality induced by B. asper venom from Costa Rica. Similar observations were performed by Theakston et al. (1995) who demonstrated that Mexican antivenom was ineective in neutralizing the most important Ecuadorian Bothrops venoms. Our results show that IB and ICP antivenoms neutralized the lethal eect of B. asper and B. jararaca venoms. It has been previously observed that these antivenoms were eective in the neutralization of a variety of heterologous snake venoms (Theakston et al., 1995, GutieÂrrez et al., 1996). To judge the cross-neutralization of an heterologous venom by a particular antivenom, it is important to evaluate not only the neutralization of lethality, but also of other relevant toxic activities (GutieÂrrez et al., 1990, 1996). For instance, a monospeci®c anti-B. lanceolatus antivenom neutralized lethality and hemorrhagic activity of B. asper and B. atrox venoms, but failed to neutralize coagulant and de®brinating eects (BogarõÂ n et al., 1999). On the basis of the cross-neutralization observed in this study it would be relevant to perform a more comprehensive evaluation of the neutralizing ability of IB and ICP antivenoms against several medically-relevant Bothrops sp venoms in Latin America. Acknowledgements This work was supported by FAPESP, European Community (CI1CT94-0043), Fundac° aÄo Butantan, CNPq and VicerrectorõÂ a de InvestigacioÂn, Universidad de Costa Rica (project 741-89-057).
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GutieÂrrez, J.M., 1995. Clinical toxicology of snakebite in Central America. In: Meir, J., White, J. (Eds.), Handbook of Clinical Toxicology of Animal Venoms and Poisons. CRC Press, Boca Raton, Florida, pp. 645±665. GutieÂrrez, J.M., Rojas, G., BogarõÂ n, G., Lomonte, B., 1996. Evaluation of the neutralizing ability of antivenoms for the treatment of snakebite envenoming in Central America. In: Bon, C., Goyon, M. (Eds.), Envenomings and their Treatments. Fondation Marcel MeÂrieux, Lyon, pp. 223±231. Klinman, N.R., Rockey, J.H., Karush, F., 1965. Equine antihapten antibody. II. The G (7 S-g) components and their speci®c interaction. Immunochemistry 2, 51±60. Kondo, H., Kondo, S., Ikezawa, I., Murata, R., Ohsaka, A., 1960. Studies of the quantitative method for determination of hemorrhagic activity of habu snake venom. Jpn. J. Med. Sci. Biol. 13, 43±51. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Mebs, D., Polhmann, S., Von Tenspolde, W., 1988. Snake venom hemorrhagins: Neutralization by commercial antivenoms. Toxicon 26, 435±438. MinisteÂrio da SauÂde, 1990. Acidentes ofõÂ dicos: Contribuic° aÄo ao estudo da morbidade. Secretaria de Ac° oÄes BaÂsicas de SauÂde, BrasõÂ lia. Powrie, F., Coman, R.L., 1993. Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunology Today 14, 270±274. Raw, I., Guidolin, R., Higashi, H.G., Kelen, E.M.A., 1991. Antivenins in Brazil: Preparation. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Vol 5, Reptile Venoms and Toxins. Marcel Dekker, New York, pp. 557±581. Rosenfeld, G., 1971. Symptomatology, pathology and treatment of snake bites in South America. In: BuÈcheri, W., Buckley (Eds.), Venomous Animals and their Venoms, Vol II, Venomous Vertebrates. Academic Press, New York, pp. 346±379. Russell, F.E., 1988. Snake venom immunology: Historical and practical considerations. J. Toxicol Ð Toxin Rev. 7, 1±82. Stephano, M.A., Yamaguchi, I.K., Hisako, H.G., Sant'Anna, O.A., 1996. Depletory activity of Lachesis muta venom on the antibody response to unrelated antigens. In: IV SimpoÂsio da Sociedade Brasileira de Toxinologia, Recife, Brasil. Theakston, R.D.G., Lloyd-Jones, M.J., Reid, H.A., 1977. Micro-ELISA for detecting and assaying snake venom and anti-venom antibody. Lancet ii, 639±641. Theakston, R.D.G., Reid, H.A., 1983. Development of simple standard assay procedures for the characterization of snake venoms. Bull. WHO 61, 949±956. Theakston, R.D.G., Warrell, D.A., 1991. Antivenoms: a list of hyperimmune sera currently available for the treatment of envenoming by bites and stings. Toxicon 29, 1419±1470. Theakston, R.D., Laing, G.D., Fielding, C.M., Lascano, A.F., Touzet, J.M., Vallejo, F., Guderian, R.H., Nelson, S.J., Wuster, W., Richards, A.M., 1995. Treatment of snake bites by Bothrops species and Lachesis muta in Ecuador: laboratory screening of candidate antivenoms. Trans. R. Soc. Trop. Med. Hyg. 89, 550±554. Towbin, H., Staehelin, T., Gordon, J., 1979. Eletrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350±4354.
Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica I. Fernandes a,*, E.X. Lima a, H.A. Takehara a, A.M. Moura-da-Silva a, I. Tanjoni a, J.M. GutieÂrrez b a
LaboratoÂrio de Imunopatologia, Instituto Butantan, SaÄo Paulo, Brazil Instituto Clodomiro Picado, Facultad de MicrobiologõÂa, Universidad de Costa Rica, San JoseÂ, Costa Rica
b
Received 18 May 1999; accepted 6 July 1999
Abstract Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000±000. This work compared the speci®city, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys ) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus ) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by anity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG pro®le and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic
* Corresponding author at: LaboratoÂrio de Imunopatologia, Instituto Butantan, Av. Vital Brazil 1500, 05503-900, SaÄo Paulo, Brazil. Fax: +55-11-815-1505. E-mail address: [email protected] (I. Fernandes). 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 7 7 - 4
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antivenom of Instituto Butantan being more eective. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: IgG isotypes; Cross-neutralization; Snake antivenoms; Horse
1. Introduction Snakes of the genus Bothrops are widespread in Latin America, and are responsible for most snakebites in this region (MinisteÂrio da SauÂde, 1990; Fan and Cardoso, 1995; GutieÂrrez, 1995). In Brazil, species of Bothrops cause more than 70% of accidents by snakebites (MinisteÂrio da SauÂde, 1990) and Bothrops jararaca is involved in many of them. On the other hand, B. asper is the most important species in Central America from a medical point of view (BolanÄos, 1982, GutieÂrrez, 1995). Bothrops sp envenomations are characterized by in¯ammation, edema and extensive local tissue damage at the site of the bite, together with systemic eects such as hemorrhage, coagulopathies, renal failure and cardiovascular shock (Rosenfeld, 1971; GutieÂrrez and Lomonte, 1989; Fan and Cardoso, 1995). Antivenoms obtained from hyperimmunized horse plasmas constitute the only speci®c treatment for snakebite envenomation. Despite long-term experience with antivenom therapy, relatively little is known about horse antivenom IgG isotypes. It is of relevance to know the IgG isotype pro®le of antivenoms as well as the role of each isotype in toxin neutralization, since ideally only the eective isotypes should be administered to patients. Horse IgG isotypes have been designated IgGa, IgGb, IgGc and IgG(T), based on their immunoelectrophoretic mobility (Klinman et al., 1965). In order to clarify the role of major isotypes present in bothropic and crotalic antivenoms from Instituto Butantan, IgG(T) and IgGa have been isolated and their neutralizing ability evaluated (Fernandes et al., 1991, 1994 and 1997). These two isotypes contained most of the antivenom speci®c antibodies responsible for the neutralization of lethality and other important venom activities. Antivenoms are currently produced in many laboratories around the world (Theakston and Warrell, 1991), with very dierent immunization protocols and methodologies for immunoglobulin puri®cation. Two of the major producers in Latin America are the Instituto Butantan in Brazil and Instituto Clodomiro Picado in Costa Rica. The former produces a bothropic antivenom by immunizing horses with venoms of ®ve species of Bothrops sp predominant in the southeastern Brazil (Raw et al., 1991) whereas Instituto Clodomiro Picado produces a polyvalent antivenom with an immunization mixture containing venoms of Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys (BolanÄos and Cerdas, 1980). It is therefore important to study if the dierences in the type of venoms used for immunization, as well as in the protocols followed, result in antivenoms with dierent neutralizing capacities and IgG isotype pro®le.
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The present study compared antibody titers, speci®city and IgG subclass content of the polyvalent antivenom of Instituto Clodomiro Picado and the bothropic antivenom of Instituto Butantan which are used in these countries in the treatment of Bothrops sp envenomations. In addition, the major isotypes IgG(T) and IgGa were isolated from both antivenoms and a comparative analysis was conducted on their neutralizing ecacy against lethal, coagulant, hemorrhagic and phospholipase A2 activities of the venoms. Finally, the cross neutralization of both antivenoms against B. jararaca and B. asper venoms was also evaluated. 2. Materials and methods 2.1. Animals Two-month-old male Swiss mice, weighing 18±22 g, were used. 2.2. Venoms B. jararaca venom was extracted from snakes held at the Instituto Butantan and B. asper venom from specimens kept at Instituto Clodomiro Picado, Costa Rica. Venoms were lyophilized and stored at ÿ208C. 2.3. Antivenoms The polyvalent antivenom of Instituto Clodomiro Picado (ICP) (anti-B. asper, C. d. durissus and L. m. stenophrys ) and the bothropic antivenom of Instituto Butantan (IB) (anti-B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus ) were used throughout. Immunization protocols and adjuvants used have been previously described (Raw et al., 1991; Angulo et al., 1997). Goat antihorse IgG(T) was purchased from ICN Immunobiologicals (Lisle, IL, U.S.A.) and rabbit anti-horse IgGa, IgGb, IgGc were purchased from Bethyl Laboratories INC (Montgomery, ALA, U.S.A.). 2.4. Isolation of horse IgG(T) and IgGa from antivenom IgGa and IgG(T) isotypes were isolated from IB or ICP antivenoms by chromatography on protein A-Sepharose followed by chromatography on a column of anti-horse IgG(T) monoclonal antibody (LO-HoGT-1)-Sepharose as reported by Fernandes et al. (1994). Brie¯y, a 2.5 cm 8.0 cm protein ASepharose column was equilibrated with 0.15 M borate buered saline, pH 8.0 (BBS) and 0.5 ml of ICP or IB antivenom was applied to the column which was washed with BBS until no protein could be detected in the euent. The bound IgG isotypes were then eluted with 0.15 M citrate buer pH 5.5. All procedures were performed at 48C. Immunochemical analysis of the material eluted at pH 5.5 using rabbit anti-horse immunoglobulins showed that it contained only IgG(T)
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and IgGa. Separation of the isotypes was then achieved by passing the eluted mixture in the column of Sepharose-LO-HoGT-1. The ®rst peak obtained by ¯ushing the column with BBS contained IgGa whereas the second peak, eluted with 0.15 M citrate buer pH 5.0, contained IgG(T). The purity of these fractions was evaluated by immunoelectrophoresis. The IgG(T) and IgGa isotypes isolated from horse IB or ICP antivenom were then used to ®nd out their ability to neutralize lethality of B. jararaca or B. asper venoms, respectively. 2.5. Antibody assay (ELISA) The antibody titers of the dierent IgG subclasses of IB and ICP antivenoms to B. jararaca or B. asper venoms were determined by ELISA according to the method of Theakston et al. (1977), with some modi®cations. Brie¯y, 50 mg/ml venom were used to coat the plates and IB or ICP antivenom dilutions were added followed by 1 mg/ml goat anti-horse IgGa, IgGb, IgGc or IgG(T). Next, peroxidase labeled anti-goat Ig6 (Sigma) was added followed by orthophenylene diamine plus H2O2 as substrate. Absorbances were recorded in an ELISA reader (Multiskan spectrophotometer, EFLAB, Helsinski, Finland) and the titers were determined as the reciprocal of the highest dilution that causes an absorbance greater than 0.100 at 492 nm, as non-speci®c reactions were observed below this value. 2.6. Determination of LD50 The i.p. LD50 for B. jararaca (33 mg/20 g mouse) and for B. asper (77.5 mg/20 g mouse) venoms were determined by probit test (Finney, 1971) using groups of six mice at each dose. Survival was recorded 48 h after venom injection. The data were analyzed according to Almeida et al. (1990). 2.7. Neutralization of lethality Neutralizing ability of ICP or IB antivenom is expressed as eective dose 50% (ED50), de®ned as microlitres of antivenom per milligram of venom at which half of the injected animals survived (GutieÂrrez et al., 1990). The challenge dose of venom used was three LD50. The ED50 for IgGa and IgG(T) is de®ned as milligram of protein per milligram of venom. Samples were mixed with venom dissolved in saline. The mixtures were incubated for 30 min at 378C, centrifuged to remove the immune complexes and the supernatant injected i.p. into mice. Control animals received the same amount of venom dissolved in the same volume of saline without antibodies. Survival was determined 48 h later. 2.8. Antihemorrhagic activity The method of Kondo et al. (1960), as modi®ed by GutieÂrrez et al. (1985), was used. Brie¯y, a constant amount of venom was incubated with various dilutions of
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antibodies, in order to obtain several antibody/venom ratios. Mixtures were incubated for 30 min at 378C and aliquots of 0.1 ml containing 10 minimum hemorrhagic doses (MHD, amount of venom that induces a hemorrhagic lesion of 10 mm diameter 2 h after injection) were injected i.d. in the abdominal region of groups of four Swiss mice. Control mice were injected with either venom alone or antibodies alone. Two hours after injection animals were sacri®ced, their skin removed and the hemorrhagic areas in the inner side of the skin measured. Neutralization was expressed as ED50, de®ned as the ratio mg antibody/mg venom in which the eect of venom alone was neutralized by 50%. 2.9. Neutralization of coagulant activity The method of Theakston and Reid (1983), as modi®ed by Gene et al. (1989) was followed, using human citrated plasma. A constant amount of venom was incubated with various dilutions of antibodies. Mixtures were incubated as described above. Then, 0.1 ml of the mixtures, containing two minimum coagulant doses of venom (MCD, minimal amount of venom that induces coagulation of plasma in 60 s) were added to samples of 0.2 ml of citrated plasma and the clotting times recorded. In control tubes plasma was incubated with either venom alone or antibodies alone. Neutralization was expressed as eective dose (ED), de®ned as the ratio mg antibodies/mg venom at which the clotting time increased three times when compared with clotting time of plasma incubated with two MCD of venom alone (Gene et al., 1989). 2.10. Neutralization of PLA2 activity (indirect hemolysis) The method described by GutieÂrrez et al. (1988) was used. Brie¯y, a constant amount of venom was incubated with various dilutions of antibodies and mixtures were incubated as described before. Then, aliquots of 10 ml of the mixtures were added to wells in agarose-egg yolk-sheep erythrocyte gels (GutieÂrrez et al., 1988). Control samples included venom incubated without antibodies and antibodies incubated without venom. Plates were incubated at 378C for 20 h and neutralization expressed as the ratio mg antibodies/mg venom able to reduce by 50% the diameter of the hemolytic halo when compared to the eect induced by venom alone. 2.11. Western blotting Venom antigens were separated by electrophoresis according to the method of Laemmli (1970) using 8 to 18% polyacrylamide gel plus SDS (SDS-PAGE) under non-reducing conditions. The gels were either stained with 0.2% (w/v) Coomassie Brilliant Blue G-250 in methanol:water:acetic acid (4:5:1) or transferred at 100 V for 2 h to a 0.45 mm pore size nitrocellulose membrane using the Pharmacia (Uppsala, Sweden) Trans-Blot system by the procedure of Towbin et al. (1979). The nitrocellulose membrane was cut into strips and incubated for 2 h in PBS
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plus 5% defatted milk to block unoccupied sites. All incubations and washing steps were performed on an end-to-end rotor at room temperature. The strips were incubated with a 1:5,000 dilution of IB or ICP antivenom for 1 h. Some strips were incubated with conjugated rabbit anti-horse IgG peroxidase and developed while others were incubated with goat anti-horse IgGa, IgGb, IgGc or IgG(T) followed by rabbit anti-goat IgG and then developed by the addition of freshly prepared 4a-chloro-1-a-naphtol at 3 mg/ml plus hydrogen peroxide. The reaction was allowed to proceed for 1±5 min and stopped by washing strips with water. Lysozyme 14,300, b-lactoglobulin 18,300, carbonic anhydrase 29,000, ovalbumin 46,900, bovine serum albumin 68,000, phosphorylase B 97,400 and myosin-H 200,000 were used as mol. wt markers. 3. Results IB and ICP antivenoms are produced under dierent immunization schedules using as antigens the predominant Bothrops species in Brazil and Costa Rica, respectively. First, we compared the antibody titer, speci®city and neutralizing ability of both antivenoms when confronted with either the homologous venom (used in the immunization protocol of the antivenom in test) or the heterologous one (present in the immunization protocol of the other antivenom). Despite the dierences in the immunization protocol, both antivenoms presented similar antibody titers against the homologous venom when assayed by ELISA, the same being observed for their IgG isotypes (Table 1). Interestingly, high antibody titers were also detected when whole antivenoms were assayed using heterologous venoms. In these cases, the dierences in antibody titers were around two dilution steps (Table 1). The IgG isotypes were also similar for both IB and ICP antivenoms: IgG(T) is the major isotype for antivenom antibodies, followed by IgGa, with smaller titers for IgGb and IgGc (Table 1). The speci®city of ICP and IB antivenoms was also compared. B. asper and B. jararaca venoms dier considerably in their electrophorectic pattern with the Table 1 Antibody titers of IB and ICP antivenoms and their dierent IgG subclasses to B. jararaca and B. asper venomsa
Whole antivenom IgG(T) IgGa IgGb IgGc
IB (B. jararaca )
IB (B. asper )
ICP (B. asper )
ICP (B. jararaca )
1,024,000 1,024,000 64,000 16,000 16,000
1,024,000 512,000 256,000 64,000 32,000
512,000 512,000 128,000 32,000 8,000
256,000 128,000 64,000 16,000 4,000
a The antibody titers were determined by ELISA as the reciprocal of the highest dilution with an absorbance greater than 0.100 at 492 nm, since non-speci®c reactions were observed below this value.
Fig. 1. Western blotting analysis of whole antivenoms (A) and IgG isotypes of IB (B) and ICP (C) antivenoms. Approximately 5 mg of B. asper (Ba) or B. jararaca (Bj) venoms were resolved by SDS-PAGE using 8 to 18% under non-reducing conditions and either stained by Coomassie blue (cb) or transferred to nitrocellulose membrane. After blocking unoccupied sites the membrane was incubated with IB or ICP antivenom, followed by rabbit anti-horse IgG peroxidase conjugate or goat anti-horse IgGa (a), IgGb (b), IgGc (c) or IgG(T) (T) followed by rabbit anti-goat IgG conjugate. Arrows on the left indicate the migration of mol. wt markers.
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predominance of low mol. wt. bands in the former and high mol. wt. bands in the latter (Fig. 1(A), lines Ba/cb and Bj/cb). Despite that, ICP and IB antivenoms recognized the same pattern by immunoblotting in each venom (Fig. 1(A)). The speci®city of the antibodies contained in each isotype was also consistent. There was no dierence in the ability of each IgG subclass to recognize venom antigens. However, IgG(T) gave a stronger reaction, followed decreasingly by IgGa, IgGb and IgGc, these results being in agreement with the antibody titers observed by ELISA. The same results were observed for IB (Fig. 1(B)) and ICP (Fig. 1(C)) antivenoms. The neutralizing capacity of both antivenoms for homologous and heterologous venoms was also determined. As shown in Table 2, ICP and IB antivenoms had a similar ability to neutralize homologous venoms. However, the polyvalent antivenom of ICP neutralized the heterologous B. jararaca venom less eciently than that of B. asper. Signi®cant dierences were also detected in the neutralization of B. jararaca venom by IB and ICP antivenoms, the ®rst being more ecient. Interestingly, the neutralization of B. asper venom was achieved similarly by homologous and heterologous antivenoms (Table 2). The IgGa and IgG(T) isotypes were isolated from both antivenoms by a combination of two anity chromatographic procedures, protein A-Sepharose followed by anti-IgG(T) mAb (LO-HoGT-1)-Sepharose and tested against the homologous venoms. The neutralizing ability of these subclasses is also shown in Table 2. IgG(T) was always more eective than IgGa from IB or ICP antivenoms. In IB antivenom, the relative concentration of neutralizing antibodies is at least 3 times higher in IgG(T) than in IgGa. In ICP antivenom, neutralizing antibodies were almost exclusively of IgG(T) isotype (Table 2). Table 2 Ability of IB and ICP antivenoms and their IgG(T) and IgGa subclasses to neutralize lethality in mice induced by B. jararaca and B. asper venomsa B. jararaca IB Antivenomb
B. asper
90.1 (70.5±109.6) 4.1 (3.74.4) 13.3 (9.7±18.7)
83.8 (76.6±90.5) ND
IgG(T)c
284.7 (222.2±331.3) ND
IgGac
ND
114.6 (74.8±141.6) 4.6 (4.0±5.3) > 13.0
IgG(T)c IgGab ICP Antivenomb
ND
a The neutralizing ability was expressed as the ED50, de®ned as microliters of antivenoms b or milligrams of isolated immunoglobulin isotypes c per milligram of venom. ND: not determined.
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Table 3 Neutralization of hemorrhage, coagulation and indirect hemolysis induced by B. asper and B. jararaca venoms using IgG(T) and IgGa isolated from IB and ICP antivenomsa
Hemorrhage Coagulation Indirect hemolysis a
Neutralization of B. jararaca venom by IB antivenom
Neutralization of B. asper venom by ICP antivenom
IgG(T)
IgGa
IgG(T)
IgGa
4.420.5 5.320.4 4.120.5
12.421.1 11.221.2 12.321.4
1.920.1 12.020.8 28.023.0
8.120.7 16.0 20.9 > 40.0
Neutralization of the activities is expressed as mean2SD (mg antibody/mg venom).
IgGa and IgG(T) isotypes were also tested regarding their ability to neutralize hemorrhagic, coagulant and PLA2 (indirect hemolytic) activities. The results of these experiments are shown in Table 3. In both antivenoms IgG(T) was the major isotype responsible for neutralization of these activities, followed by IgGa. 4. Discussion Although antivenoms derived from hyperimmune horse plasma have been used for over a century in the treatment of snakebite envenomation, little is known about the content of various IgG isotypes in these immunobiologicals, as well as on the neutralizing ability of the dierent isotypes. Antivenoms produced in Brazil and Costa Rica are used in several Latin American countries, and eorts are being made to gain a better knowledge and understanding of antivenom characteristics and neutralizing pro®le. Despite important dierences in the immunization protocols used at Instituto Clodomiro Picado and Instituto Butantan, as well as in the venoms used, both antivenoms presented similar antibody titers and speci®cities for the various IgG isotypes studied. Nevertheless, slight dierences were noticed, since IgGa from ICP antivenom failed to neutralize phospholipase A2 activities and lethality induced by B. asper venom. Several factors may control the levels of each isotype during the immune response. In mice and humans, the restriction of antibody isotype response is associated with activation of speci®c subpopulations of Thelper cells which present a dierent pattern of cytokine expression (Powrie and Coman, 1993). However, little is know about isotype control in horse immune response. Some components in the venoms of C. d. terri®cus and L. m. muta have been associated with alterations in the immune response in mice (Cardoso and Mota, 1997; Stephano et al., 1996). Crotoxin, the main protein of C. d. terri®cus venom is a potent immunesupressor, aecting the proliferation of lymphocytes and the cytokine balance (Cardoso et al., 1998). Immunosupression was also observed with L. m. muta venom when mice were immunized with various antigens
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(Stephano et al., 1996). Interestingly, the presence of Crotalus and Lachesis venoms in the immunizing mixture of polyvalent antivenom did not seem to alter the titer or the isotype pro®le of the product. However, the presence of these components may be related to the reduction in the neutralizing ability of IgGa. This suggestion is supported by the observation that anti-crotalic IgGa is seven times less potent than IgG(T) while anti-bothropic IgGa is only three times (Fernandes et al., 1997). Regarding the neutralizing ability of the isotypes, IgG(T) was more ecient than IgGa, on a weight per weight basis, in the neutralization of lethal, hemorrhagic, coagulant and indirect hemolytic activities. This agrees with previous ®ndings of Fernandes et al. (1997) and con®rms that IgG(T) is the most important immunoglobulin in bothropic and crotalic antivenoms. On this basis, some manufacturers have introduced chromatographic steps in their production protocols aimed at the puri®cation of IgG(T), with the consequent reduction in total protein concentration of antivenoms while keeping a high neutralizing titer (Grandgeorge et al., 1996). Our observations showed a strong cross-neutralization of IB and ICP antivenoms when confronted with heterologous venoms. Immunological crossreactivity in venom±antivenom systems has been well documented (Mebs et al., 1988; Russell, 1988; Ferreira et al., 1992; GutieÂrrez et al., 1996; de Roodt et al., 1998). In contrast, BogarõÂ n et al. (1995) described that three heterologous antivenoms produced in dierent Latin American countries failed to neutralize lethality induced by B. asper venom from Costa Rica. Similar observations were performed by Theakston et al. (1995) who demonstrated that Mexican antivenom was ineective in neutralizing the most important Ecuadorian Bothrops venoms. Our results show that IB and ICP antivenoms neutralized the lethal eect of B. asper and B. jararaca venoms. It has been previously observed that these antivenoms were eective in the neutralization of a variety of heterologous snake venoms (Theakston et al., 1995, GutieÂrrez et al., 1996). To judge the cross-neutralization of an heterologous venom by a particular antivenom, it is important to evaluate not only the neutralization of lethality, but also of other relevant toxic activities (GutieÂrrez et al., 1990, 1996). For instance, a monospeci®c anti-B. lanceolatus antivenom neutralized lethality and hemorrhagic activity of B. asper and B. atrox venoms, but failed to neutralize coagulant and de®brinating eects (BogarõÂ n et al., 1999). On the basis of the cross-neutralization observed in this study it would be relevant to perform a more comprehensive evaluation of the neutralizing ability of IB and ICP antivenoms against several medically-relevant Bothrops sp venoms in Latin America. Acknowledgements This work was supported by FAPESP, European Community (CI1CT94-0043), Fundac° aÄo Butantan, CNPq and VicerrectorõÂ a de InvestigacioÂn, Universidad de Costa Rica (project 741-89-057).
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