Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248190

Conclusions: It appears according to this study that thisimmune serum could be a great contribution to cover allthe polymorphisms existing in our country.

Table 1Effect of tri-valent antibodies (F(ab’)2 anti Aah, anti Amx and anti Bot) on metabolical perturbations induced by scorpion venoms.

CPK (IU/l) AST (IU/l) ALT (IU/l) ALP (IU/l) Urea (mmol/l) Crea (mmol/l)

Control 596.5 � 29.8 87 � 8.2 17 � 1.2 73.79 � 8 7.59 � 1 1.32 � 0.09Aah Venom 1546 � 77.3 192.6 � 15.3 56 � 5 184 � 15 10.8 � 0.9 16 � 0.8Bot Venom 1543 � 51.4 180 � 17 81.2 � 7 107 � 10 8.6 � 1 13 � 1.1Amx venom 2608 � 65.2 247.6 � 21.3 49.7 � 5 88 � 9 7.9 � 8 13 � 1.2Tri-valent-seraþ Aah venom 408 � 20.1 141.2�14 27.6 � 3.2 70 � 7 6.6 � 7.1 6 � 0.5Tri-valent-seraþ Bot venom 1098 � 36.6 163.7 � 15.3 22.7 � 1.8 68 � 7.1 8.2 � 0.95 4 � 0.3Tri-valent-seraþ Amx venom 934 � 31 132.8 � 10.2 44.6 � 5 61 � 5.9 6.8 � 0.5 12 � 1

TCPK: creatine phospho-kinase; AST: aspartate transaminase; ALT: alanine transaminase; ALP: alkaline phosphatise; Crea: creatinemia.

Fig. 1. Hepatic parenchymal and myocardial structure in envenomed miceand treated by trivalent-antibodies (Haematoxylin and eosin coloration,magnification 400, legends: H: hemorrhage, E: edema).

Keywords: antibodies, valence, scorpion venom, neutralization10.1016/j.toxicon.2012.04.184

184. Development of Novel Scorpion Anti-Venoms inMéxico

Baltazar Becerril, Lidia Riaño, Lourival D. PossaniDepartment of Molecular Medicine and Bioprocesses, Instituto deBiotecnologia, National Autonomous University of Mexico, Av. Universidad,2001, Cuernavaca, MexicoE-mail address: baltazar@ibt.unam.mx (B. Becerril).

Background: Scorpions of the family Buthidae containvenoms that provoke severe envenoming to humans. Toxiccomponents are short-chain peptides that affect ion-chan-nels (being Naþ-channels the most important), causing anabnormal cellular function. Scorpions of the genus Centrur-oides usually have few highly toxic peptides in their venoms.We have shown that a monoclonal antibody (BCF2) wascapable of neutralizing the intoxication caused by both: toxinCn2 and the whole venom from the scorpion CentruroidesnoxiusHoffmann (Licea et al. Toxicon, 34, 843-847,1996). Thisfinding prompted the hypothesis that anti-venoms againstscorpions could be constituted by a few neutralizing anti-bodies. In order to confirm this idea, a library of single-chain

human antibodies (scFv) was constructed and evaluated bymeans of phage-display and directed evolution.

Methods: A selected scFv (3F) was evolved resulting ina neutralizing antibody (6009F) of the whole solublevenom of Centruroides noxius (Riaño et al FEBS J. 272:2591-2601, 2005; Patent US 7,381,802 B2).

Results: New scFv variants like 9004G havebeen generated. We demonstrated that 6009F and 9004Gare capable of protecting mice against venoms from twodifferent scorpion species: Centruroides suffusus suffususand Centruorides noxius. A key residue (F101VH) wasinserted into scFv 9004G lone improving its neutralizingcapacity. The scFv obtained (LR) is the best neutralizingantibody against main toxins and venoms of these twoscorpion species obtained to date by our group (Riaño et al.J. Biol. Chem. 286:6143-6151, 2011).

Conclusions: These results show that a single antibodymay have multiple neutralizing capacities. Following thesame strategy, several scFvantibodies capable of neutralizingthe main toxins of Centruroides limpidus limpidus have beenobtained. These results allow us to propose confidently thata cocktail containing a few optimized human scFv antibodieswill become the next generation of scorpion anti-venoms.

AcknowledgementsThis work has been partially supported by Instituto

Bioclón, S.A. de C.V., Mexico and DGAPA-UNAM IN204110 toLDP and CONACyT to BB.

Keywords: antivenom; phage display; scorpion; directed evolution10.1016/j.toxicon.2012.04.185

185. Safety of Equine F(ab’)2 Antivenom for ScorpionEnvenomation: Results of Prospective Clinical Trials

Leslie Boyer 1, Michelle Ruha 2, Jan Degan 1, Jody Mallie 1,Alejandro Alagón 3

1Venom Immunochemistry, Pharmacology, and Emergency Response (VIPER)Institute, University of Arizona, Tucson, AZ, USA2Banner Good Samaritan Medical Center, Phoenix, AZ, USA3 Instituto de Biotecnología, Universidad Nacional Autónoma de México,Cuernavaca, MéxicoE-mail address: boyer@viper.arizona.edu (L. Boyer).

Background: Use of serum products includingantivenoms is known to involve a risk of type 1 and type 3hypersensitivity. The technology involved in the produc-tion of serum derivatives has advanced during the pastcentury such that third-generation (“fabotherapeutic”)

Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248 191

products now have much higher potency relative to totalprotein content, and the quantity of immunoreactivematerial injected into patients has been in many casesgreatly reduced. Recent prospective clinical trials of equineF(ab’)2 scorpion antivenom provided an opportunity toconduct follow-up evaluations of safety outcomes with oneof these newer products.

Methods: Children and adults at selected sites in the USand Mexico, envenomated by scorpions between 2004 and2011, were eligible for enrollment in a series of fiveprospective clinical trials of AnascorpTM (Centruroides (Scor-pion) Immune F(ab’)2 (Equine) Injection). In all protocols,adverse events were prospectively monitored, includingspecific observation for symptoms suggestive of serumsickness during the two weeks following enrollment.Adverse events were categorized as to severity and possiblecause, all serious adverse events were individually reviewed,and descriptive statistics were applied to the results.

Results: 15 patients were enrolled in a double-blindprotocol, 7 of whom received antivenom. 78 patients wereenrolled in 3 open label protocols that mirrored the treat-ment group in the double-blind trial. 1425 additionalpatients were enrolled in a treatment protocol offered at 28hospitals across Arizona. The range of antivenom exposurewas 1-5 vials, intravenous. In the treatment protocol, 307adults and 1118 children were treated. Overall, 3 (0.2%) hadacute type 1 reactions and 8 (0.6%) appeared to have type 3reactions; but none had the full syndrome of classicalserum sickness. Therewere 30 serious adverse events, mostof which involved secondary respiratory consequences ofenvenomation or of excessive sedation concomitant withthe study. There were no deaths.

Discussion: Acute and delayed immune responses area risk with any serum derivative; but results of thesestudies show that refined third-generation fabother-apeutics can have remarkably low adverse event profiles, incontrast with historic descriptions of 60-90% rates of serumsickness using first generation antisera. This study was notdesigned to prove cause, but it is likely that this improvedsafety is a consequence of high potency (thus relatively lowprotein dose), lack of immunogenic Fc with the F(ab’)2fragment, and high purity of this preparation overall.

Keywords: safety, antivenom, hypersensitivity, serum sickness, clinical trial10.1016/j.toxicon.2012.04.186

N. Snakes

186. Effects of Captivity or Season on VenomComposition in Two Species of Rattlesnakes(Crotalus atrox and C. v. viridis)

Christopher J. Rex, Stephen P. MackessyUniversity of Northern Colorado, School of Biological Sciences, Greeley,CO USAE-mail address: christopher.j.rex@gmail.com (C.J. Rex).

Background: Snake venoms consist of a variety ofbiochemical components that serve to immobilize, kill and

digest prey. Venom variability within snakes has typicallybeen attributed to factors such as age, season, environmentand diet, but studies exploring snake venom composition atfine scales of resolution (within individual snakes) areuncommon. Toexplore theeffects of several of thesevariables,two separate studies were performed: effects of captivity onthe venom composition of adult Western DiamondbackRattlesnakes (Crotalus atrox), and seasonal effects on venomcomposition in snakes collected during spring and fall (free-ranging adult Prairie Rattlesnakes, Crotalus viridis viridis).

Methods: Sixteen C. atrox were captured from CochiseCo., AZ and maintained in captivity for eight months ona diet of NSA mice. Thirty-three C. v. viridis were captured,PIT-tagged, and released in the spring and fall from twowell-defined den sites in Weld Co., CO. Venoms wereextracted shortly after capture and once every two-threemonths for the C. atrox. Venom samples from both studieswere subjected to reducing 1-D SDS-PAGE, reversed-phaseHPLC, MALDI-TOF mass spectrometry (MS), five differentenzyme assays and a fibrinogen degradation assay.

Results: For both studies, venom composition appearedto remain constant within individuals, as assessed by 1-DSDS-PAGE and fibrinogen digest assay results, while RP-HPLC and MALDI-TOF MS showed only minor differences.For C. atrox, venom L-amino acid oxidase (LAAO) andphosphodiesterase (PDE) activity significantly increasedover the course of captivity, with no changes occurring inmetalloproteinase (MPr), kallikrein-like serine protease(KLSP), or thrombin-like serine protease (TLSP) activities.Crotalus v. viridis venoms showed significantly higher PDEactivity and lower MPr activity in the spring than in the fall.Crotalus v. viridis venom TLSP activity levels also increasedsignificantly with time/age (from spring to fall and fall tospring), with no changes in LAAO or KLSP activity.

Discussion: Since the overall “fingerprint” for eachsnake's venom remained more/less constant, it can beconcluded that major changes in venom composition didnot occur within individuals in either species. Small butstatistically significant differences were, however, observedfor some venom enzyme activities.

Conclusions: These studies indicate that minor differ-ences in venom composition do occur in two rattlesnakespecies, as a function of season or captivity/diet. Althoughfurther testing will be necessary, these differences arelikely to be of minimal significance when treating cases ofhuman envenomation or producing antivenom.

Keywords: snake, venom, variation, Crotalus, atrox, viridis, season, captivity.10.1016/j.toxicon.2012.04.187

187. Metalloproteinases from Rear-Fanged (“Colubrid”)Snake Venoms: An Under-Utilized Resource forEvolutionary and Structure/Function Studies

Stephen P. MackessySchool of Biological Sciences, University of Northern Colorado, Greeley, CO, USAE-mail address: stephen.mackessy@unco.edu.

Background: Rear-fanged snakes (“Colubridae”) areabundant world-wide, and because their venom proteomesare typically much less complex than those of viperids and