©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS 8 RESEARCH ARTICLE Antivenomics of Atropoides mexicanus and Atropoides picadoi snake venoms: Relationship to the neutralization of toxic and enzymatic activities José Antúnez α , Julián Fernández α , Bruno Lomonte α , Yamileth Angulo α , Libia Sanz α , Alicia Pérez β , Juan José Calvete β , José María Gutiérrez α, * α Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica, β Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científi cas (CSIC), Jaume Roig 11, 46010 Valencia, Spain * Correspondence to: José María Gutiérrez, Email: jose.gutierrez@ucr.ac.cr , Tel: +506 2229 3135, Fax: +506 2292 0485 Submitted: 14 July 2010; Revised: 16 August 2010; Accepted: 20 August 2010; Published: 30 September 2010 J Venom Res, 2010, Vol 1, 8-17 © Copyright The Authors: This is an open access article, published under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/). This license permits non-commercial use, distribution and reproduction of the article, provided the original work is appropriately acknowledged with correct citation details. ABSTRACT Viperid snakes of the genus Atropoides are distributed in Mexico and Central America and, owing to their size and venom yield, are capable of provoking severe envenomings in humans. This study evaluated, using an ‘antivenomics’ approach, the ability of a polyspecifi c (polyvalent) antivenom manufactured in Costa Rica to recognize the proteins of Atropoides mexicanus and A. picadoi venoms, which are not included in the immu- nization mixture. In addition, the neutralization of lethal, hemorrhagic, myotoxic, coagulant, proteinase and phospholipase A 2 (PLA 2 ) activities of these venoms by the antivenom was assessed. The antivenom was highly- effective in immunodepleting many venom components, particularly high molecular mass P-III metallopro- teinases (SVMPs), L-amino acid oxidases, and some serine proteinases and P-I SVMPs. In contrast, PLA 2 s, certain serine proteinases and P-I SVMPs, and a C type lectin-like protein were only partially immunodepleted, and two PLA 2 molecules were not depleted at all. The antivenom was able to neutralize all toxic and enzymatic activities tested, although neutralization of lethality by A. nummifer venom was achieved when a challenge dose of 3 LD 50 s of venom was used, but was iffective when 4 LD 50 s were used. These results, and previously obtained evidence on the immunoreactivity of this antivenom towards homologous and heterologous venoms, revealed the low immunogenicity of a number of venom components (PLA 2 s, CRISPs, P-I SVMPs, and some serine proteinases), underscoring the need to search for innovative immunization protocols to improve the immune response to these antigens. KEYWORDS: Atropoides picadoi , Atropoides mexicanus , antivenomics , antivenom , neutralization , toxicity , immunodepletion , snake venom INTRODUCTION Animal-derived antivenoms constitute the cornerstone in the treatment of envenomings by snakebites worldwide ( WHO, 2010 ). In contrast to other antibody-based immunotherapeu- tics, such as various antitoxins and rabies immunoglobu- lin, the antigens (snake venoms) used for the preparation of snake antivenoms exhibit a large intra- and inter-species variability ( Chippaux et al, 1991 ; Alape-Girón et al, 2008 ; Calvete et al, 2010b ). Consequently, the selection of the venoms to be used in the immunizing mixtures to raise snake antivenoms is a key issue in the design of these immunobio- logicals ( Gutiérrez et al, 2009a ; WHO, 2010 ). Antivenoms can be either monospecifi c, if venom of only one species is used for immunization, or polyspecifi c, when animals are immunized with venoms from two or more species 9©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS provides a comprehensive view of the cross-reactivity and neutralization spectrum of antivenoms against homologous and heterologous venoms. In turn, this information can be used for a more rigorous design of immunizing mixtures for the manufacture of more effective antivenoms. The venomes of two species of Atropoides snakes, A. nummifer ( mexicanus ) and A. picadoi , from Costa Rica have been recently reported ( Angulo et al, 2008 ). Despite the close phylogenetic kinship of these two species, their venoms comprise rather dissimilar proteomes, with phos- pholipases A 2 (PLA 2 s) being the major toxins present in A. mexicanus venom, whereas Zn 2+ -dependent metallopro- teinases (SVMPs) predominate in A. picadoi venom ( Angulo et al, 2008 ). Such proteomic profi les are consistent with the pathophysiological alterations induced by A. mexicanus and A. picadoi venoms in mice, as the former induces prominent PLA 2 -mediated myonecrosis while the latter causes SVMP-mediated hemorrhage, being the venom with the highest hemorrhagic potential among Costa Rican viperids ( Gutiérrez et al, 1985 ). The present work is aimed at describing a detailed antivenomic assessment of the immunoreactivity against the venoms of A. mexicanus and A. picadoi of the polyspecifi c antivenom used in Central America. In addition, the neutralization of the most relevant toxic and enzymatic activities of A. mexicanus and A. picadoi venoms by the antivenom was also investigated. The results evidenced a conspicuous pattern of cross-reactivity and par- aspecifi c protection of the antivenom, but also identifi ed a number of venom components against which the antivenom has a weak antibody repertoire, thus providing useful infor- mation for the improvement of this immunotherapeutic. MATERIALS AND METHODS Venoms and antivenom Venoms were obtained from at least 20 adult specimens of each species collected in Costa Rica and kept at the ser- pentarium of Instituto Clodomiro Picado (ICP). Venoms were freeze-dried immediately after collection, and stored at -20ºC. Polyspecifi c (polyvalent, Crotalinae) antivenom (Batch 420, expiry date October 1 st , 2010) from ICP was used. This antivenom is routinely prepared at ICP from the plasma of horses immunized with a mixture of the venoms of Bothrops asper , Crotalus simus and Lachesis stenophrys ( Angulo et al, 1997 ), and consists of immunoglobulins puri- fi ed by caprylic acid precipitation ( Rojas et al, 1994 ). A con- trol preparation of normal equine IgG was prepared by an identical fractionation of the plasma of horses which had not been immunized with snake venoms. Antivenomics: Immunodepletion of venom proteins by the ICP polyvalent antivenom We have coined the term “antivenomics” for the proteomic characterization of venom proteins bearing epitopes rec- ognized by an antivenom ( Lomonte et al, 2008 ; Calvete et al, 2009a and 2009b ; Gutiérrez et al, 2009a ). Briefl y, two milligrams of whole venom were dissolved in 70µl of 20mM phosphate buffer, pH 7.0, and mixed with 8mg of polyvalent antivenom IgGs overnight at room temperature (∼22ºC). IgG concentration was determined spectrophoto- metrically at 280nm using an extinction coeffi cient (ε) of 1.4 for a 1mg/ml protein concentration determined in a 1cm ( Theakston et al, 2003 ; WHO, 2010 ). In either case, the gen- erated antibodies recognize not only proteins present in the homologous venoms, but also antigenically-related proteins present in heterologous venoms of phylogenetically-related species. Such paraspecifi city may in turn result in effective neutralization of venoms not included in the immunizing mixture, as has been shown in the case of Bothrops anti- venoms in Latin America ( Segura et al, 2010 ). However, there are also situations in which antivenoms fail to neutralize ven- oms of closely related species, as has been documented for neotropical rattlesnakes ( Saravia et al, 2002 ; Calvete et al, 2010b ). Therefore, the detailed analysis of the paraspecifi c neutralization and immunoreactivity of antivenoms against venoms of medically-relevant species is a necessary task for establishing their preclinical spectrum of effi cacy. The family Viperidae comprises 23 snake species in Cen- tral America ( Campbell and Lamar, 2004 ), some of which are responsible for the vast majority of snakebites in this region ( Gutiérrez, 2009 ). These species are classifi ed within the genera Agkistrodon, Atropoides Bothriechis, Bothrops, Cerrophidion, Crotalus, Lachesis and Porthidium ( Campbell and Lamar, 2004 ). The genus Atropoides includes a number of thick-bodied species, known as jumping vipers, distrib- uted in Mexico and Central America ( Campbell and Lamar, 2004 ). Although very little information is available regard- ing the incidence of snakebites caused by these species to humans, it is likely that they infl ict a number of accidents due to their broad distribution and relative abundance. In addition, the similarity of clinical symptoms with those caused by other pitvipers, such as Bothrops asper , makes the identifi cation of Atropoides bites rather diffi cult. Despite the lack of epidemiological records on bites by these species, their size and relatively high venom yield ( Bolaños, 1972 ) suggests that they may be able to provoke signifi cant enven- omings in humans. A polyspecifi c antivenom, prepared by immunizing horses with the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys , is widely used in Central America for the treatment of envenomations caused by species of the family Viperidae ( Angulo et al, 1997 ; Gutiérrez, 2009 ). Early on after the development of this antivenom, its ability to neutralize the lethal effect of viperid venoms from Costa Rica, including those of Atropoides species, was demon- strated ( Bolaños, 1971 ). Further studies evaluated the neu- tralization of proteolytic, hemorrhagic, indirect hemolytic, edema-forming, coagulant and defi brinating activities of Central American snake venoms by this antivenom, includ- ing the venoms of Atropoides nummifer ( mexicanus ) and A. picadoi ( Gutiérrez et al , 1985 , 1986 ; Rojas et al, 1987 , 2001 ; Gené et al, 1989 ; Valiente et al, 1992 ). Recently, pro- teomic analytical tools have been adapted for the analysis of the immunoreactivity of antivenoms against venoms, a fi eld of study coined ‘antivenomics’ ( Lomonte et al, 2008 ; Gutiérrez et al, 2009a ; Calvete et al, 2009a ; Calvete, 2010 ). Once the proteomic profi le of a particular venom (the ‘venome’) is deciphered, then the immunoreactivity of antivenoms against the different venom components can be investigated, thus allowing a detailed assessment of the immune recognition scope of antivenoms. This information, together with the analysis of the neutralizing ability against specifi c toxicological and enzymatic effects of venoms, 10 ©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS estimated as the amount of venom inducing a hemorrhagic halo of 10mm in the skin 2hr after injection. Myotoxicity was determined in mice (18-20gm) injected intramuscu- larly, in the right gastrocnemius, with solutions of varying venom concentration ( Instituto Clodomiro Picado, 2008 ). Three hours after injection, mice were bled and the plasma creatine kinase (CK; E.C. 2.7.3.2) activity was determined by means of a kinetic assay (CK-Nac, Biocon Diagnostik, Germany). The Minimum Myotoxic Dose (MMD) was defi ned as the amount of venom that induced an increment in plasma CK activity corresponding to 4 times the value in control mice injected with PBS alone. Coagulant activity was determined in human citrated plasma ( Theakston and Reid, 1983 ; Gené et al, 1989 ). The Minimum Coagulant Dose (MCD) corresponds to the amount of venom that induces clotting of plasma in 60sec. Proteolytic activity was deter- mined using azocasein as substrate ( Wang et al, 2004 ), as modifi ed by Gutiérrez et al (2008) . One unit of proteolytic activity corresponds to the amount of venom that induces a change in absorbance at 450nm of 0.2. PLA 2 activity was determined titrimetrically using egg yolk phospholipids as substrate ( Dole, 1956 ; Gutiérrez et al, 1986 ). Activity was expressed as µEq fatty acid released per mg protein per min. All experiments involving the use of animals were approved by the Institutional Committee for the Care and Use of Lab- oratory Animals (CICUA) of Universidad de Costa Rica. Neutralization of venom activities by the polyvalent antivenom was assessed by using a previously described protocol based on the incubation of a fi xed dose of venom (‘challenge dose’) with various dilutions of antivenom, for 30min at 37ºC, in order to achieve various ratios of µl antivenom/mg venom ( Gutiérrez et al, 1990 ; Instituto Clodomiro Picado, 2008 ). Then, aliquots of the mixtures, containing a challenge dose of venom, were tested in the cor- responding experimental systems described above. Controls included venom solutions incubated without antivenom. The challenge doses of venom used were; lethality, 3 or 4 LD 50 ; hemorrhagic activity, 10 or 5 MHD; myotoxic activ- ity, 3 MMD in the case of A. picadoi and 5 MMD in the case of A. nummifer ; coagulant activity, 3 MCDs; proteolytic activ- ity, 10µg for both venoms; and PLA 2 activity, 80µg venom for A. nummifer and 40µg for A. picadoi. The challenge doses for proteolytic and PLA 2 activities were selected from dose-response curves and corresponded to doses exert- ing submaximal activity. Neutralization was expressed as Effective Dose 50% (ED 50 ), corresponding to the ratio µl antivenom/mg venom, which reduced by 50% the effect induced by the venom ( Gutiérrez et al, 1990 ). In the case of coagulant activity, neutralization was expressed as Effec- tive Dose (ED), corresponding to the ratio µl antivenom/mg venom in which the clotting time of plasma was prolonged three times when compared with the clotting time of plasma incubated with venom alone. RESULTS Antivenomics Figures 1 and 2 , and Table 1 present the results of the immuno depletion of venom proteins by the antivenom. The antivenom completely immunodepleted various pro- teins of A. mexicanus venom (C-toxins), immunodepleted 60-70% of the major PLA 2 s 21 and 22, and 75-80% of light path length cuvette ( Fasman, 1992 ). Thereafter, the reaction mixture was incubated for 1hr at 37ºC with 400µl of Protein G-Sepharose (nominal binding capacity of 20mg IgG/ml) (GE-Healthcare) in 20mM phosphate buffer, pH 7.0. Protein G-bound immunocomplexes were sedimented at 3,000rpm for 10min in an Eppendorf microcentrifuge, and the supernatant was submitted to reverse-phase HPLC separation using an ETTAN TM LC HPLC system (Amer- sham Biosciences) and a Lichrosphere RP100 C 18 column (250x4mm, 5µm particle size) developed at 1ml/min with a linear gradient of 0.1% (w/v) TFA in water (solution A) and acetonitrile (solution B) (5%B for 10min, followed by 5-15%B over 20min, 15-45%B over 120min, and 45-70%B over 20min) ( Angulo et al, 2008 ). Protein detection was at 215nm and peaks were collected manually and dried in a Speed-Vac (Savant). HPLC-fractions were assigned to pre- viously characterized toxins ( Angulo et al, 2008 ) by their HPLC elution profi le, N-terminal sequencing (using a Procise instrument, Applied Biosystems, Foster City, CA, USA) and SDS-PAGE. Control samples were subjected to the same procedure except that control equine IgGs, instead of antivenom IgGs, were included in the reaction mixture. Quantitation of immunodepletion was assessed by compar- ing the peak areas of control and treated samples. Depend- ing on the extent of immunodepletion, venom proteins were categorized as: C-toxins, corresponding to comple- tely immunodepleted proteins; P-toxins, corresponding to partially immunodepleted proteins; and N-toxins, non- immunodepleted proteins ( Calvete et al, 2009b ). Antivenomics: Immunoblot analysis The presence of polyvalent antivenom antibodies direc- ted against antigenic determinants of A. mexicanus and A. picadoi venom proteins was also investigated by Western Blot analysis. To this end, the reverse-phase HPLC chro- matographic fractions were separated by SDS-PAGE (15%, w/v, polyacrylamide concentration) under non-reducing conditions, followed by electrotransfer to nitrocellulose membranes ( Towbin et al, 1979 ) using a Bio-Rad minitrans- fer cell operated at 150mA during 90min. To assess trans- fer effi ciency, nitrocellulose membranes were visualized by reversible Ponceau-S Red staining. Then, the membranes were destained by exhaustive washing with distilled water, and incubated in 2% (w/v) bovine serum albumin in phos- phate-buffered saline solution (PBS; 0.12M NaCl, 0.04M sodium phosphate, pH 7.2) for 30min at room temperature to block non-specifi c binding sites, followed by incubation for 90min with 1:3,000 dilution (in PBS) of antivenom or control equine IgGs. After washing four times with PBS containing 0.2% (w/v) albumin and 0.05% (v/v) Tween-20, the membranes were incubated for 90min with anti- horse IgG-alkaline phosphatase conjugate (Sigma) diluted 1:6,000. Finally, the membranes were washed fi ve times as above, and color development was performed by adding BCIP/NBT (Chemicon) substrate. Neutralization of toxic and enzymatic activities The toxic and enzymatic activities of the venoms were assessed using previously described protocols. Briefl y, lethality was determined by intraperitoneal injection in CD-1 mice (16-18gm) ( Bolaños, 1972 ). Hemorrhage was assessed by the intradermal test in mice ( Gutiérrez et al, 1985 ). The Minimmum Hemorrhagic Dose (MHD) was 11 ©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS of A. picadoi that were not recognized by immunoblot were proteins 16 (PLA 2 ), 17 (CRISP), 18 (PLA 2 ), a P-I SVMP present in fractions 25, 26 and 28, and a C type lectin-like protein eluted in fraction 30 ( Figure 3 and Table 1 ). In this venom, a number of proteins that were immunodepleted by the antivenom failed to be recognized by immunoblotting ( Table 1 ). On the other hand, PLA 2 15 and serine proteinase 24 of A. picadoi venom were not immunodepleted, but were recognized by antivenom IgGs in western blot ( Table 1 ). Neutralization of toxic and enzymatic activities Table 2 displays the toxic and enzymatic activities of the venoms of A. mexicanus and A. picadoi . In the case of proteolytic activity on azocasein, it largely depends on the action of SVMPs, since incubation of venoms with 20 mM EDTA impaired by > 95% the activity of both venoms. On the basis of these activities, the challenge doses described in the Materials and Methods section were selected. The serine proteinases 26, 27, 28, 31 and 32 (P-toxins), and did not reduce the peak of PLA 2 23 (N-toxin) ( Figure 1 and Table 1 ). On the other hand, most proteins were quantitatively immunodepleted from the venom of A. picadoi by the antivenom ( Figure 2 and Table 1 ), thus corresponding to C-toxins. However, proteins 24 (a serine proteinase) and 26 (a P-I SVMP) were only partially (∼40%) depleted from the venom, thus corresponding to P-toxins ( Table 1 ). Nota- bly, PLA 2 15 was not immunoprecipitated at all (N-toxin). Immunoblot analysis was in line with most of the results obtained by the immunodepletion approach ( Figure 3 ). In the case of A. mexicanus venom, N- and P-toxins were also poorly recognized by Western blotting, with the exception of fractions 26 and 27 (serine proteinases). A further dis- crepancy between immunoblotting and immunodepletion in A. mexicanus venom was on protein 24 (a cysteine-rich secretory protein, CRISP), which was not recognized by the antivenom in Western blot but was partially immunode- pleted in the antivenomic assay. Proteins from the venom Figure 1. Reverse-phase HPLC separation of proteins from control ( A ) and polyvalent antivenom- immunodepleted ( B ) Atropoides mexicanus venom. Protein number- ing and identity correspond to those in Table 1 of Angulo et al (2008) . Letters correspond to fractions con- taining IgG molecules. The inserts show a photo of an adult A. picadoi specimen and a SDS-PAGE analysis of the β-mercaptoethanol-reduced fractions isolated by reverse-phase HPLC. Venom proteins are labeled with asterisks. 12 ©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS lethal activity of venoms in mice ( WHO, 1981 ). Although the mouse lethality test still remains as the gold standard for antivenom potency estimation ( WHO, 2010 ), the biochemi- cal and toxicological complexity of snake venoms urges a more comprehensive analysis of antivenom preclinical effi cacy. A relevant step forward in this area was the intro- duction of neutralization tests based on specifi c toxic and enzymatic venom activities besides lethality ( Theakston, 1986 ; Gutiérrez et al, 1996 ; Instituto Clodomiro Picado, 2008 ). In the case of viperid snake venoms, the neutraliza- tion of hemorrhagic, coagulant, defi brinogenating and myo- toxic activities are of particular relevance. Recently, a novel approach in the assessment of antivenom immunoreactiv- ity, termed ‘antivenomics’, has been developed ( Lomonte et al, 2008 ; Gutiérrez et al, 2009a ; Calvete et al, 2009a ). The antivenomic approach is based on a proteomic methodologi- cal platform, which permits the identifi cation of all proteins present in venoms, allowing therefore the analysis of the immunoreactivity of an antivenom against each individual component in a venom. Immunoblotting techniques have polyvalent antivenom was effective in the neutralization of the toxic and enzymatic activities of the two Atropoides venoms ( Table 3 ). Concerning the lethal activity of A. mexi- canus venom, the antivenom failed to neutralize this effect when a challenge dose of 4 LD 50 s was used. However, the antivenom neutralized lethality when the challenge dose was 3 LD 50 s. In contrast, the lethal activity of A. picadoi venom was readily neutralized by the antivenom even when using a challenge dose of 4 LD 50 s ( Table 3 ). Neutralization of hem- orrhagic and PLA 2 activities required, respectively, the least and the highest volume of antivenom ( Table 3 ). For com- parative purposes, the neutralizing ability of this antivenom against the toxic and enzymatic effects of Bothrops asper venom, which is used in the immunizing mixture for the preparation of this antivenom, is also shown in Table 3 . DISCUSSION Preclinical assessment of the neutralizing ability of antiven- oms has been traditionally based on the neutralization of the Figure 2. Reverse-phase HPLC separation of proteins from control ( A ) and polyvalent antivenom- immunodepleted ( B ) Atropoides picadoi venom. Protein numbering and identity corresponds to those in Table 2 of Angulo et al (2008) . Letters correspond to fractions containing IgG molecules. The insert shows a photo of an adult A. mexicanus specimen and a SDS-PAGE of the β-mercaptoethanol-reduced fractions isolated by reverse-phase HPLC. Venom proteins are labeled with asterisks. 13 ©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS proteinase, a P-I SVMP and a C-type lectin-like protein. There were two PLA 2 s – one in each venom – which were not recognized by the antivenom, being therefore N-toxins. The toxic profi le of these poorly recognized proteins remains unknown. Our fi ndings resemble previ- ous results obtained with the venoms of Bothrops asper ( Gutiérrez et al, 2010 ), B. caribbaeus and B. lanceolatus ( Gutiérrez et al, 2008 ), Bothriechis lateralis and B. schlegelii ( Lomonte et al, 2008 ). In these cases, high molecular mass venom proteins, such as P-III SVMPs and L- amino acid oxidases, were completely immunodepleted by the antivenom (C-toxins), whereas proteins of lower molecu- lar mass, especially disintegrins, CRISPs and PLA 2 s were immunodepleted only to a partial extent (P-toxins) or, in some cases, not depleted at all (N-toxins). Other compo- nents of intermediate molecular mass, such as serine pro- teinases, P-I SVMPs and C-type lectin-like proteins showed an intermediate pattern of immunodepletion. The fact that a clear relationship exists between molecular mass of venom proteins and their immunorecognition by antivenom IgGs regardless of their relative abundance in the venoms is not surprising, since the surface area accessible to the immune system is directly proportional to the molecular mass of the protein. Hence, the contact area of antigen and anti- body varies between 600 and 1200 A 2 and involves about 14-21 surface-exposed residues ( Padlan et al, 1989 ), and thus a small protein binds simultaneously less IgG mol- ecules than a higher molecular mass protein. Interestingly, the low immunoreactivity of the polyvalent antivenom against a number of Atropoides venom toxins appears not to depend on the fact that these venoms were not included in the immunizing mixture, since a highly similar pattern of immunodepletion was observed against the venom of B. asper ( Gutiérrez et al, 2010 ), which is included in the venom mixture used in immunization ( Angulo et al, 1997 ). been also used for identifying venom proteins recognized by antivenom antibodies ( Lomonte et al, 2008 ; Correa-Netto et al, 2010 ). The combination of immunodepletion, immu- noblotting, and neutralization approaches allows a detailed evaluation of the preclinical effi cacy of antivenoms. Here we use this integrated methodology to uncover the paraspe- cifi c reactivity and neutralization ability of a polyvalent antivenom against the venoms of two species of Atropoides from Central America. A modifi cation of the original antivenomics protocol was introduced for this work. Instead of precipitating venom-antivenom complexes by the addition of anti-horse IgG, as originally described by Lomonte et al (2008) , the immune complexes were removed by adsorption to protein G-Sepharose beads, which bind horse IgG Fc domains, thereby allowing their depletion from the reaction mix- ture by centrifugation. This modifi cation greatly reduces the concentration of free anti-horse IgG molecules in the supernatant, thus simplifying the reverse-phase chroma- tographic separation. As in previous antivenomic studies ( Lomonte et al, 2008 ; Gutiérrez et al, 2008 ; 2010 ), this approach was combined with western blot analysis of antivenom reactivity. In general, immunoprecipitation and immunoblotting analyses had a good correlation, with some exceptions. Such discrepancies may be based on the fact that immunoblotting is performed on denatured proteins, which may affect some conformational epitopes in venom compo- nents. The antivenom recognized most of the proteins in the two Atropoides venoms by both antivenomic immunode- pletion and immunoblotting. However, a number of toxins were immunodepleted only to a partial extent. In the case of A. mexicanus venom, P-toxins included PLA 2 s, CRISP, and various serine proteinases. Similarly, the A. pica- doi venom’s P-toxins comprised PLA 2 s, CRISP, a serine Species Fractions Molecular mass (Da) Techniquea Protein Family A. mexicanus 21 13,751 WB and IP PLA2 (Myotoxin I) 22 13,792 WB and IP PLA2 (Myotoxin II) 24 24,744 WB CRISP 26 30,000 IP Serine proteinase 27 27 26,000 33,000 IP IP Serine proteinase Serine proteinase 28 26,000 WB and IP Serine proteinase 31 34,000 WB and IP Serine proteinase 32 34,000 WB and IP Serine proteinase A. picadoi 15 13,897.6 IP PLA2 16 13,806.6 WB PLA2 17 24,787.1 WB CRISP 18 13,790.6 WB PLA2 24 28,000 IP Serine proteinase 25 22,900 WB P-I SVMP 26 22,900 WB and IP P-I SVMP 28 22,900 WB P-I SVMP 30 28,000 WB C type lectin-like Table 1. Proteins from the venoms of A. mexicanus and A. picadoi that were only partially immunodepleted or not recognized by immunoblotting with polyvalent antivenom. Numbering of fractions corresponds to those of Figures 1 and 2. aTechnique by which immunoreactivity was assessed: Western Blot (WB) or immunoprecipitation (IP). 14 ©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS Figure 3. Antivenomic profi le of A. mexicanus ( A ) and A. picadoi ( B ) snake venoms by western blot analysis. Venom fractions isolated by HPLC were then separated by SDS-PAGE under non-reducing conditions, and transferred to nitrocellulose membranes. Ponceau-S Red reversible staining of transferred RP-HPLC fractions is shown at left and western blot of the fractions is shown at right. Lane numbers correspond to peaks in HPLC chromatography, and migra- tion of molecular mass markers is depicted to the left. Circles are used to highlight protein bands not recognized by the polyvalent antivenom. Some bands not recognized by the antivenom were not included in Table 1 because their identity has not been determined in proteomic analysis. 15 ©The Authors | Journal of Venom Research | 2010 | Vol 1 | 8-17 | OPEN ACCESS there is a signifi cant antigenic similarity between the main toxins in B. asper and Atropoides venoms, and that the low immunoreactivity against some venom components in Atropoides is not due to the heterologous nature of these venoms but, instead, to the low immunogenicity of some venom components, i.e. , PLA 2 s, CRISPs, and some P-I SVMPs and serine proteinases. The proteomic differences between the venoms of A. mexicanus and A. picadoi have evident implications in the neutralization by antivenom. P-III SVMPs comprise 56% of the venom of A. picadoi ( Angulo et al, 2008 ). P-III SVMPs are mostly responsible for the hemorrhagic activ- ity of venoms ( Gutiérrez et al, 2005 ) and, in the case of A. picadoi , it is likely that P-III SVMPs constitute major lethal factors and also toxins responsible for local myo- toxicity, since this venom has a low content of myotoxic PLA 2 s ( Angulo et al, 2008 ); consequently, local myonecro- sis is mostly secondary to the massive microvessel dam- age, leading to ischemia induced by hemorrhagic SVMPs. Since P-III SVMPs are readily immunodepleted by poly- valent antivenom, this explains the high effi cacy of this anti venom to neutralize the hemorrhagic, myotoxic and lethal effects of A. picadoi venom. In contrast, in the case of A. mexicanus venom, neutralization of hemorrhagic activity is highly effi cient whereas neutralization of lethality requires a higher volume of antivenom. Since P-III SVMPs comprise only 3.4 % of the venom of A. mexicanus ( Angulo et al, 2008 ), it is suggested that the lethality of this venom by the intraperitoneal route is probably due to the action of PLA 2 s. This is in line with the relatively low toxicity of the venom, i.e. , high LD 50 , since myotoxic PLA 2 s from Central Ameri- can viperid venoms are lethal only at relatively high doses ( Gutiérrez and Lomonte, 1995 ; Gutiérrez and Lomonte, 1997 ). Hence, the different proportions of P-III SVMPs and PLA 2 s in these venoms largely explain the variations in the neutralization of lethality. On the other hand, the neutraliza- tion of the proteolytic activity on azocasein by the Costa Rican polyvalent antivenom is 31 and 12 times less effective than the neutralization of the hemorrhagic activity of the Neutralization studies confi rmed the effi cacy of the poly- valent antivenom to neutralize the toxic and the enzymatic activities of the two Atropoides venoms, albeit in the case of lethality induced by A. mexicanus venom, such neutraliza- tion was achieved only when a challenge dose of up to 3 LD 50 s was used. Our fi ndings agree with previous studies on the neutralization of Atropoides venoms by this polyspecifi c antivenom, although quantitative differences in the values of ED 50 s have been reported, probably evidencing varia- tions between different batches of antivenom or intraspecies variability in the composition of venoms from various geo- graphical origins ( Gutiérrez et al, 1985 ; Rojas et al, 1987 ; 2001 ; Gené et al, 1989 ; Bogarín et al, 2000 ). As in the case of immunodepletion experiments, the ED 50 values for the neutralization of toxic and enzymatic activities of these heterologous venoms were similar to those for the neutra- lization of B. asper venom, which is part of the immuniz- ing mixture ( Saravia et al, 2001 ; Gutiérrez et al, 2010 ; and Table 3 ). Moreover, in the case of neutralization of PLA 2 activity, antivenom was more effective against heterolo- gous Atropoides venoms than against homologous B. asper venom ( Table 3 ). This further supports the concept that Activitya A. mexicanus A. picadoi Lethality (LD 50 ) 125μg 42μg Hemorrhagic (MHD) 6μg 0.25μg Myotoxic (MMD) 4.2μg 13µg Coagulant (MCD) 15μg No coagulation at 50μg Proteolytic (units/mg) 150 145 Phospholipase A 2 (μEq/mg/min) 4.6 11.6 Table 2. Toxic and enzymatic activities of the venoms of A. nummifer and A. picadoi. aToxic activities are expressed as Median Lethal Dose (LD 50 ), Minimum Hemorrhagic Dose (MHD), Minimum Myotoxic Dose (MMD) and Minimum Coagulant Dose (MCD) (See Materials and Methods for details). Neutralization (ED 50 )a Venom activity A. mexicanus A. picadoi Bothrops asperb Lethality (4 LD 50 s) >2000µl/mg 353 (249-500) µl/mg 342 (224-465) Lethality (3 LD 50 s) 891 (565-1404) µl/mg Not tested Not tested Hemorrhagic 112 ± 27µl/mg 48 ± 38µl/mg 175 ± 37µl/mg Myotoxic 1134 ± 275 380 ± 174 555 ± 159 Coagulant 972 ± 36µl/mg Venom devoid of coagulant effect 133 ± 8µl/mg Proteolytic 1394 ± 44µl/mg 1481 ± 36µl/mg 942 ± 22µl/mg Phospholipase A 2 402 ± 16µl/mg 901 ± 69µl/mg 1268 ± 182µl/mg Table 3. Neutralization of toxic and enzymatic activities of A. mexicanus and A. picadoi venoms by the polyvalent antivenom from Instituto Clodomiro Picado. aNeutralization is expressed as the Effective Dose 50% (ED 50 ) corresponding to the ratio µL antivenom/mg venom at which the effect of the challenge dose of venom was reduced to 50%. In the case of coagulant effect, neutralization is expressed as Effective Dose (ED), corresponding to the ratio µL antivenom/mg venom at which the clotting time of plasma is prolonged three times as compared with clotting time of plasma incubated with venom alone (see Materials and methods for details). In the case of lethality, the 95% confi dence limits are given in parentheses. For the other effects, results are presented as mean ± S.D. (n = 4). bResults of neutralization of B. asper venom correspond to those described by Saravia et al (2001) using similar methodologies. 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Hemor- rhage induced by snake venom metalloproteinases: Biochemical venoms of A. picadoi and A. mexicanus , respectively, despite the fact that both effects are due to SVMPs, as judged by the ability of EDTA to abolish them. Such apparent discrep- ancy is likely due to the fact that the hemorrhagic activity is largely due to P-III SVMPs whereas the proteolytic activity is mainly dependent on the action of P-I SVMPs, which in general have a higher enzymatic activity, albeit being less toxic, than P-III SVMPs ( Gutiérrez et al, 2009b ). Since P-I SVMPs are immunodepleted to a lesser extent than P-III SVMPs by the antivenom, antivenomic and neutralization results clearly match regarding the neutralization of hemor- rhagic and proteolytic effects. CONCLUSIONS • Our observations demonstrate an extensive cross- neutralization of A. mexicanus and A. picadoi venoms by the polyvalent antivenom used for the treatment of viperid envenomings in Central America. • Antivenomic observations evidenced that antivenoms readily immunodeplete P-III SVMPs from both ven- oms, but immunodepletion of PLA 2 s, CRISPs and some P-I SVMPs and serine proteinases was achieved only to a partial extent, thus revealing the scarcity of (high-affi nity) antibodies against these proteins. • These fi ndings, together with previous antivenomic studies, stress the need to search for immunization strategies aimed at enhancing the immune response against these poorly immunogenic venom toxins in order to increase the effi cacy and neutralizing potency of antivenoms. ACKNOWLEDGEMENTS This study was supported by projects from the Vicerrec- toría de Investigación, Universidad de Costa Rica (741-A7- 611), CRUSA-CSIC (2007CR0004), CONARE, and grants BFU2007-61563 and BFU2010-17373 from the Ministerio de Ciencia e Innovación, Madrid, Spain. STATEMENT OF COMPETING INTERESTS None declared. REFERENCES Alape-Girón A, Sanz L, Escolano J et al. 2008. Snake venomics of the lancehead pitviper Bothrops asper : geographic, individual and ontogenetic variations. J Proteome Res 7, 3556-3571. Angulo Y, Estrada R and Gutiérrez, JM. 1997. Clinical and labora- tory alterations in horses during immunization with snake ven- oms for the production of polyvalent (Crotalinae) antivenom. Toxicon, 35, 81-90. Angulo Y, Escolano J, Lomonte B, Gutiérrez JM, Sanz L, Calvete JJ. 2008. Snake venomics of Central American pitvi- pers. Clues for rationalizing the distinct envenomation profi les of Atropoides nummifer and Atropoides picadoi . J Proteome Res, 7, 708-719. 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