Sphingomyelinase D from venoms of Loxosceles spiders: evolutionary insights from cDNA sequences and gene structure.

Loxosceles spider venoms cause dermonecrosis in mammalian tissues. The toxin sphingomyelinase D (SMaseD) is a sufficient causative agent in lesion formation and is only known in these spiders and a few pathogenic bacteria. Similarities between spider and bacterial SMaseD in molecular weights, pIs and N-terminal amino acid sequence suggest an evolutionary relationship between these molecules. We report three cDNA sequences from venom-expressed mRNAs, analyses of amino acid sequences, and partial characterization of gene structure of SMaseD homologs from Loxosceles arizonica with the goal of better understanding the evolution of this toxin. Sequence analyses indicate SMaseD is a single domain protein and a divergent member of the ubitiquous, broadly conserved glycerophosphoryl diester phosphodiesterase family (GDPD). Bacterial SMaseDs are not identifiable as homologs of spider SMaseD or GDPD family members. Amino acid sequence similarities do not afford clear distinction between independent origin of toxic SMaseD activity in spiders and bacteria and origin in one lineage by ancient horizontal transfer from the other. The SMaseD genes span at least 6500bp and contain at least 5 introns. Together, these data indicate L. arizonica SMaseD has been evolving within a eukaryotic genome for a long time ruling out origin by recent transfer from bacteria.     

Primary culture of venom glands from the Brazilian armed spider, Phoneutria nigriventer (Araneae, Ctenidae).

Phoneutria spider venoms are a rich source of bioactive components. The limited amounts of crude material available, however, can be considered as a major hindrance for a faster development in the field. In the present study, we attempted to establish primary cultures of venom glands of Phoneutria nigriventer as an alternative, in vitro source of venom. Three different developmental stages were tried as starting materials: whole embryo (inside the cocoon), nymph (early after cocoon hatching) and young adult (1 year after cocoon hatching). The embryonic cells remained in suspension in the primary cultures, with no signs of adhesion or differentiation, for about 6 months. Nevertheless, this culture was useful for the first chromosome C-banding of Phoneutria. An average of 29+/-1 acrocentric chromosomes were found. Striated muscle cells were the only kind of cells in the culture of venom glands from Phoneutria nymphs. The most promising results were achieved with 1-year-old specimens. Besides muscle, adherent epithelial cells were also obtained in culture. Although these cells remained in culture for a short time (up to 48 h) immunochemical analysis of the culture supernatant evidenced the presence of Phoneutria venom components. This can be considered as a first step toward the functional cultures of venom glands of Phoneutria spiders.     

Ontogenetic changes in Phoneutria nigriventer (Araneae, Ctenidae) spider venom.

Venom-yield and composition of differently sized individuals of the medically most important Brazilian spider Phoneutria nigriventer (Keyserling, 1891) was analysed. During growth the venom-mass increases according to a fourth order function of the prosoma size, which mainly reflects an increase of the venom gland volume. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed increasing percentages of proteins < or = 17 kDa from 4.1% in the smallest analysed spiders (2-3 months-old) to 79.1% in adult female venom. Additionally, high-pressure liquid-chromatography showed an increase of a single ('main') peak from 4.6 to 64.9%, while the overall number of other major-peaks decreased. Venom from young instars completely lacked lethality in mice up to a dose of 3.28 mg/kg i.v. as compared to a LD(50) of 0.63 mg/kg for adult female or 1.57 mg/kg for adult male venom that we reported previously. In conclusion, ontogenetic changes in venom protein-composition of growing P. nigriventer are suggested to produce increasing lethality in vertebrates.     

North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab')(2) preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.     

Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')2 extrusion mechanism from blood to tissues.

Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')(2) extrusion mechanism from blood to tissues. We measured pharmacokinetic parameters for T. discrepans venom in rams. Forty, 75 or 100 microg/kg venom were injected subcutaneously in the inner side of the thigh. Plasma venom content (venenemia) was determined by enzyme-linked immunosorbent assay (ELISA) from 0 to 300 min after injecting venom. Venenemia was fit to a three-compartment model (inoculation site, plasma and extra vascular extracellular space), it was assumed that the venom may also be irreversibly removed from plasma. Calculated time course of venom content shows that at any time no more that 30% of the venom is present in plasma. Venenemia peaks at 1h and decays afterwards. Fluorescently labelled antivenom [horse anti-TityusF(ab')(2) or fraction antigen binding, immuglobulin without Fc chain covalently bound to fluorescine or fluorescamine] pharmacokinetics was determined. Although F(ab')(2) molecular weight is >/=10 times bigger that toxin's, the rate of outflow of F(ab')(2) from blood to tissues was approximately 4 times faster than the venom's outflow. Venom content in the injection site decays exponentially for >6h, this prediction was confirmed immunohistochemically. Only approximately 5% of the venom is eliminated in 10h; approximately 80% of the venom is in the tissues after 2h and remains there for >10h.     

Structural analysis of the venom glands of the armed spider Phoneutria nigriventer (Keyserling, 1891): microanatomy, fine structure and confocal observations

Spiders belonging to the genus Phoneutria (Perty, 1833), most commonly known as 'armed' spiders, are among the most dangerous species in Brazil due to high toxicity of their venom, associated with their habit of invading domestic or specific areas such as banana plantations. The venom of Phoneutria spiders is secreted by a pair of venom glands located inside their cephalothoraxes and connected to the chelicerae by two independent ducts. In the present study, the microanatomy and histological structure of the venom glands of Phoneutria nigriventer (Keyserling, 1891) were examined in detail by histochemical and conventional stains with laser confocal, scanning and transmission electron microscopies. The analysis confirmed the bulbous-shaped organ previously observed by others. The venom glands of P. nigriventer are covered externally by a double layer of striated muscles, which are arranged in a spiral fashion. This disposition of the external muscle fibers might provide the contraction movement of the venom gland to release their contents during a sting aggression. The presence of pore-like openings between the muscle fibers that cover the venom glands of P. nigriventer was considered quite remarkable. The presence of axon-like structures between the muscle fibers seen in the gland surface was also quite remarkable. The secretory epithelium of P. nigriventer invaginates into the gland lumen, contributing to the increase of the secretory surface area and also accommodating a higher number of secretory cells. Our observation of histological sections and SEM showed that the secretory cells in the venom gland of P. nigriventer form complex structures, secretory units, which originate at the base near the muscular layer and that extend into the central area until the gland lumen. Our study also identified a possible holocrine secretory mechanism of P. nigriventer venom gland, at least in the first venom milking, since we were able to see nuclei stained on confocal laser microscopy. However, our observation cannot disregard other possible types of secretory pathways in subsequent milkings, since we found no nuclei in the second and in the third venom secretions.     

Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas.

Loxosceles spiders have a wide distribution in the temperate and tropical regions of the world. Loxoscelism is characterized by necrotic skin ulceration at the bite site and, less commonly, a systemic illness that may be fatal. The purpose of this study was to characterize and compare aspects of the major medically important Loxosceles spider venoms in a standardized manner, particularly considering their neutralization by two Brazilian antivenoms. By SDS-PAGE (12% acrylamide), Loxosceles deserta, Loxosceles gaucho, Loxosceles intermedia, Loxosceles laeta and Loxosceles reclusa venoms had similar electrophoretic profiles, with the major protein bands of 32-35 kDa. All venoms exhibited gelatinolytic, caseinolytic and fibrinogenolytic activities in vitro with a large array of proteases, mainly between 18.1 and 31.8 kDa. Most of these enzymes were metalloproteases as this activity was abolished by 1,10-phenanthroline. Hyaluronidase activity was detected in a protein band of approximately 44 kDa in all venoms. Sphingomyelinase activity was demonstrated in all five venoms. Antigenic cross-reactivity, by Western blotting, was also observed among all venoms studied using commercial equine antivenoms produced in Brazil (Institute Butantan and CPPI). These antivenoms recognized mainly components between 25 and 40 kDa in all venoms with several minor components of >89 kDa. Strong cross-reactivity was also seen among all venoms through the ELISA technique (titre range: 64,000-512,000). All venoms (5 microg doses) induced a similar local reaction when injected intradermally into the flank of rabbits, demonstrating dermonecrosis, hemorrhage, vasoconstriction, edema, and erythema. However, no reaction was observed when each venom was pre-incubated (1 h, 37 degrees C) with Brazilian commercial sera prior to injection. The antivenoms also abolished the sphingomyelinase activity in vitro, suggesting the venoms of the major medically important Loxosceles spider species have generally similar toxic and enzymatic characteristics. Thus, as Brazilian commercial antivenoms are able to neutralize the dermonecrosis induced by Loxosceles venoms of diverse geographical origin, clinical studies should be undertaken on the potential for a single global Loxosceles antivenom.     

Isolation, molecular cloning and functional characterization of a novel beta-toxin from the Venezuelan scorpion, Tityus zulianus.

Sting in children by Tityus zulianus scorpions (western Venezuela) often produces cardiorespiratory arrest and death by pulmonary oedema. To assess its toxicity, lethality in mice of T. zulianus soluble venom was determined. Toxin composition was studied by fractionating the crude venom through reversed-phase HPLC. The most abundant peptide, Tz1, was purified further and its N-terminal sequence, amino acid composition and molecular mass (by electron-spray ionization mass spectrometry) determined. In the presence of Tz1, activation of recombinant rat skeletal muscle sodium channels (Na(V)1.4) was shifted about 35 mV in the hyperpolarizing direction in a prepulse-dependent manner. This typical beta-toxin effect had an apparent EC50 of 3.5 microM A cDNA sequence encoding Tz1 was isolated from T. zulianus venom gland RNA using a combination of 5'- and 3'-RACE PCR. Analysis of the encoded sequence indicated that Tz1 is the processed product of a precursor containing: (i) a 20-residue long leader peptide; (ii) the amino acid sequence of the mature toxin (64 residues); and (iii) an extra Gly-Lys tail at the C-terminus, probably removed post-translationally. A comparison of Tz1 with Tityus serrulatus beta-toxin Ts1 revealed that some of the non-conservative replacements in Tz1 lie in regions potentially involved in receptor recognition.     

Tityus perijanensis González-Sponga (Scorpiones, Buthidae): molecular assessment of its geographical distribution and venom lethality of Venezuelan populations.

An extensive field survey allowed us to expand the geographical distribution of the scorpion Tityus perijanensis in the Perijá range, western Zulia State, Venezuela, including areas where adult cases of severe scorpionism have been reported. 16S ribosomal RNA (rRNA) gene sequencing, DL(50) determination, and native PAGE suggest low genetic and venom proteomic divergence across the distribution range. The results also indicate phylogenetic divergence between T. perijanensis and T. discrepans, the species prevalent in northcentral Venezuela. T. perijanensis venom lethality (0.91-0.94 mg/kg) is comparable to that of the Brazilian T. serrulatus and ranks highest among toxic Venezuelan Tityus studied so far. The data indicate that the Perijá range should be included amongst the endemic areas of scorpionism of Venezuela and Colombia.     

Coagulant effects of black snake (Pseudechis spp.) venoms and in vitro efficacy of commercial antivenom

The coagulant effects of Australasian black snakes (Pseudechis spp.) are poorly understood and differ to the procoagulant venoms of most dangerous snakes in Australia. This study aimed to investigate in vitro coagulant effects of Pseudechis venoms and the efficacy of commercial black snake antivenom (BlSAV), tiger snake antivenom (TSAV) and specific rabbit anti-snake IgG to neutralise these effects. Using a turbidimetric assay, all six Pseudechis venoms had anticoagulant activity, as well as phospholipase A₂ (PLA₂) activity. Inhibition of PLA₂ activity removed anticoagulant effects of the venoms. Pseudechis porphyriacus was unique and had procoagulant activity independent of PLA2 activity. Both BlSAV and TSAV completely inhibited the coagulant and PLA2 activity of all Pseudechis venoms. PLA2 activity was also inhibited completely by p-Bromophenacyl bromide (pBPB) and partially by specific anti-N. scutatus IgG antibodies. Anti-N. scutatus IgG also completely inhibited anticoagulant activity of Pseudechis venom. All Pseudechis venoms showed immunological cross reactivity with specific anti-snake IgG antibodies to P. porphyriacus, Pseudechis australis and Notechis scutatus. Pseudechis venoms have in vitro anticoagulant activity that appears to be attributable to PLA₂ activity. Both antivenoms inhibited anticoagulant and PLA₂ activity at concentrations below those occurring in patients treated with one vial of antivenom. There was cross-neutralisation of Pseudechis venoms and N. scutatus antibodies that might be attributable to immunological similarities between the venoms.     

Neutralization of venoms from two Southern Pacific Rattlesnakes (Crotalus helleri) with commercial antivenoms and endothermic animal sera.

The Southern Pacific Rattlesnake (Crotalus helleri) is found in southwestern California (USA), southward through north Baja California (MX) into the northern part of southern Baja California (MX). In this study, the venoms from two Southern Pacific Rattlesnakes were characterized. The two venoms were different in color, concentration, and enzyme activities. Two commercial antivenoms neutralized both C. helleri venoms differently. Antivipmyn (Fab2H) and CroFab (FabO) neutralized both venoms but had different ED50. Four times more Fab2H antivenom was required to neutralize the C. helleri venom No. 011-084-009 than the venom from the snake No. 010-367-284. The hemorrhagic activity of two C. helleri venoms were neutralized differently by endothermic animal sera having a natural resistance to hemorrhagic activity of snake venoms. Opossums and Mexican ground squirrel sera did not neutralize the hemorrhagic activity of the venom No. 010-367-284. The sera of gray woodrats and hispid cotton rats neutralized all hemorrhagins in both C. helleri venoms. This is the first reported case in which opossum serum has not neutralized hemorrhagic activity of pit viper venom. Differences in the compositions of C. helleri venoms and their ability to be neutralized may help explain why snakebites are a difficult medical problem to treat and why effective polyvalent antivenoms are difficult to produce.     

The in vitro effects of two chirodropid (Chironex fleckeri and Chiropsalmus sp.) venoms: efficacy of box jellyfish antivenom.

The pharmacological and biochemical isolation of cnidarian venoms has been hindered by difficulties with both extracting pure venom from nematocysts and venom stability. The development of a new technique to extract active, pure venom of Chironex fleckeri and Chiropsalmus sp. has enabled identify both neurotoxic and myotoxic activity in their venoms. These activities are similar, but not identical in each species. Venom (50 micro g/ml) from both species significantly inhibited indirect and direct twitches of the chick biventer nerve-muscle preparation. Pre-incubation with 1U/ml box jellyfish antivenom did not have any significant effect on venom-induced reductions of indirect twitches. However, this activity was markedly attenuated by prior addition of 5U/ml antivenom, albeit to a lesser degree for Chiropsalmus sp. In contrast, prior addition of 5U/ml box jellyfish antivenom did not neutralise the myotoxic activity of C. fleckeri venom (50 micro g/ml), although it did inhibit the myotoxicity produced by Chiropsalmus sp. venom (50 micro g/ml). Antivenom (5U/ml) added 1h after the addition of C. fleckeri venom (50 micro g/ml) had no effect on the indirect or direct twitches of the skeletal muscle preparation. However, it partially restored the reduction in indirect twitch height caused by Chiropsalmus sp. venom (50 micro g/ml). Myotoxicity was confirmed in muscle preparations stained with hematoxylin and eosin.Therefore, although antivenom was able to neutralize the neurotoxic effects of both species, and the myotoxic effects of Chiropsalmus sp., when added prior to venom, it was unable to reverse the effects after venom addition. This suggests that antivenom is unlikely to be useful in the treatment of neurotoxic or myotoxic effects in patients, although these effects are rarely seen clinically.     

Experimental evidence that oral secretions of northwestern ring-necked snakes (Diadophis punctatus occidentalis) are toxic to their prey.

Ring-necked snakes (Diadophis punctatus) are suspected of being venomous because their Duvernoy's gland secretions have high levels of phospholipase activity, which is characteristic of many viperid and elapid venoms, and because anecdotal reports of feeding behavior are consistent with the use of a venom. We tested the toxicity of northwestern ring-necked snake oral secretions to a natural prey species, northwestern garter snakes (Thamnophis ordinoides), by injecting 2-35 microl of oral secretions intraperitoneally. All doses were 100% lethal within 180 min. The dose significantly affected the time to loss of a righting response. Neither injection of saline nor denatured oral secretions resulted in loss of a righting response or any visible detrimental effects. We suggest that northwestern ring-necked snakes may have evolved venom to subdue larger prey items than the snake would otherwise be capable of taking.     

Cytotoxic, thrombolytic and edematogenic activities of leucurolysin-a, a metalloproteinase from Bothrops leucurus snake venom.

Leucurolysin-a (leuc-a), a 23kDa non-hemorrhagic metalloproteinase, is found in venom of the viper Bothrops leucurus. Here, we examine the biological consequences of leuc-a, including thrombolytic activity, direct effects on endothelial cells in culture and edematogenic activity in vivo. We demonstrate fibrinolytic activity of leuc-a, in which the protease specifically degrades alpha, beta, and gamma-gamma chains. While not causing hemorrhaging, leuc-a does cause thrombolytic activities in whole blood clots. Endothelial cells are highly resistant to leuc-a in culture. Cell viability suffered only when cells were exposed to large quantities of the protease. Nevertheless, leuc-a induces changes in cell morphology. The impact of leuc-a on cell adhesion was confirmed by an adhesion assay, in which cell adhesion to fibronectin decreased due to leuc-a. This mild cellular impact is unlike that of crude venom, where lower concentrations triggered cell death and a greater reduction in cell adhesion. Also, leuc-a increased microvessel permeability with marked edema in mice peritoneum and foot pads. These effects are similar to those of other P-I class SVPMs. These in vivo effects were weaker when crude venom was tested. In conclusion, albeit not showing significant hemorrhagic activity, leuc-a can induce a prominent edema which appears to be significant in the local effects observed after B. leucurus venom accidents.     

Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom.

Scorpion venoms are a rich source of enzymes. Some of the enzymes such as phospholipase A2, proteolytic enzymes and phosphodiesterase are well characterized. However, hyaluronidase has not been studied extensively. In this paper we describe the purification and characterization of hyaluronidase (Hyaluronate lyase, E.C.3.2.1.35) from the Palamneus gravimanus scorpion venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The optimal pH and temperature for its maximum activity of the isolated enzyme were 4.5 and 37 degrees C, respectively, and its K(m) was 47.61 microg/ml at 37 degrees C and its specific activity was 6411.7 +/- 117TRU/min per mg against 250 +/- 4.0 TRU/min per mg for the whole desiccated venom suggesting 25-fold purification. The molecular weight of the isolated enzyme was 52 +/- 1 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography on Sephadex G-75. The enzyme was stable for 30 days in the presence of NaCl; no loss of activity was observed up to 37 degrees degrees C and showed a sharp decrease in its activity at 40 degrees C. Heparin inhibited the enzyme activity.     

Venomic analyses of Scolopendra viridicornis nigra and Scolopendra angulata (Centipede, Scolopendromorpha): shedding light on venoms from a neglected group.

Centipedes are venomous arthropods responsible for a significant number of non-lethal human envenomations. Despite this, information about the composition and function of their venom contents is scarce. In this study, we have used a 'structure to function' proteomic approach combining two-dimensional chromatography (2D-LC), electrospray ionization quadrupole/time-of-flight mass spectrometry (ESI-Q-TOF/MS), N-terminal sequencing and similarity searching to better understand the complexities of the venoms from two Brazilian centipede species: Scolopendra viridicornis nigra and Scolopendra angulata. Comparisons between the LC profiles and the mass compositions of the venoms of the two species are provided. The observed molecular masses ranged from 3019.62 to 20996.94Da in S. viridicornis nigra (total: 62 molecular masses) and from 1304.73 to 22639.15Da in S. angulata (total: 65 molecular masses). Also, the N-termini of representatives of 10 protein/peptide families were successfully sequenced where nine of them showed no significant similarity to other protein sequences deposited in the Swiss-Prot database. A screening for insecto-toxic activities in fractions from S. viridicornis venom has also been performed. Six out of the 12 tested fractions were responsible for clear toxic effects in house flies. This work demonstrates that centipede venoms might be a neglected but important source of new bioactive compounds.     

Cardiovascular effects and lethality of venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rats.

Haemodynamic effects of saline-extracted venom from nematocysts isolated from Chiropsalmus quadrigatus (Habu-kurage) were studied in anaesthetized rats. Intravenous administration of venom (0.2-5 microg protein/kg) produced immediately dose-dependent hypertension and bradycardia. Femoral blood flow transiently increased but calculated femoral vascular conductance decreased. Changes caused by 1 microg/kg of venom were reproducible, and were not affected by prazosin, atropine or BQ123 (ET(A) receptor antagonist) but were significantly attenuated by nicardipine. At doses over 2 microg/kg, hypotension and a decrease in pulse pressure were observed subsequent to transient hypertension. In 5 of 8 rats received 5 microg/kg venom and 6 of 6 rats at 10 microg/kg, death due to irreversible cardiac arrest occurred within 30 min after intravenous injection. However, during nicardipine infusion, venom (10 microg/kg) exerted only modest effects and the rats survived. Heating venom (50 degrees C for 10 min) before injection practically abolished the haemodynamic effects of 10 microg/kg venom, indicating its thermolability. Data show that C. quadrigatus venom has both vasoconstrictor and cardiodepressive effects in rats, and suggest that a calcium channel blocker can protect against the cardiovascular and lethal effects of the venom.     

Comparative study of the efficacy and safety of two polyvalent, caprylic acid fractionated [IgG and F(ab′)2] antivenoms, in Bothrops asper bites in Colombia

The efficacy and safety of two polyvalent horse-derived antivenoms in Bothrops asper envenomings were tested in a randomized, double-blind, clinical trial performed in Colombia. Both antivenoms were manufactured from the same pool of hyperimmune plasma. Antivenom A was made of F(ab′)₂ fragments, generated by pepsin digestion and caprylic acid precipitation, whereas antivenom B consisted of whole IgG molecules produced by caprylic acid precipitation followed by ion-exchange chromatography. Besides the different nature of the active substance, antivenom B had higher protein concentration, slightly higher turbidity and aggregate content. No significant differences were observed in the efficacy of antivenoms. Both halted local and systemic bleeding (P = 0.40) within 6-12 h of treatment in 100% of the cases, and restored blood coagulation (P = 0.87) within 6-24 h in 84.7% of patients, and within 48 h in all of them, in agreement with restoration of plasma fibrinogen concentration. Venom concentrations in serum dropped significantly (P < 0.001), to very low levels, 1 h after antivenom infusion. Nevertheless, eight patients (11.1%), four for each antivenom, presented recurrence of venom antigenaemia at different times, from 6 to 96 h, with clinical significance (recurrent coagulopathy) only in one group B patient (2.9%). Serum creatine kinase (CK) activity was increased, as a consequence of local myonecrosis. There was no significant difference (P = 0.51) in the incidence of early adverse reactions to antivenom administration (28.9% for patients of group A and 20.6% for patients of group B), most of the reactions being mild, mainly cutaneous. The most frequent complications were cellulitis (16.7%), abscess formation (5.6%), acute renal failure (8.3%), and compartmental syndrome (5.6%). In conclusion, IgG and F(ab′)₂ antivenoms, prepared by caprylic acid fractionation, presented similar efficacy and safety profiles for the treatment of B. asper envenomings in Colombia.     

Engineering of a recombinant Fab from a neutralizing IgG directed against scorpion neurotoxin AahI, and functional evaluation versus other antibody fragments.

Antibody-based therapy is the only specific treatment for scorpion envenomation. However, there are still major drawbacks associated with its use; mainly because antivenoms are still prepared from immune equine serum raised against crude venoms, whereas only a limited number of neurotoxins are responsible for the lethality of the venom. Using a murine hybridoma that secretes a well-characterized neutralizing IgG directed to neurotoxins AahI and AahIII from the venom of the scorpion Androctonus australis, we constructed a recombinant Fab (rFab) fragment, which was produced and purified from transformed bacteria. It recognized toxin AahI with a high affinity (KD = 8.2 x 10(-11)) equivalent to the homologous pFab prepared by papain digestion of whole IgG. Although the AahI-neutralizing capacity of protein L-purified rFab was low compared to other recombinant antibody formats (scFv and diabody) investigated in parallel, the antibody engineering approach presented here provides an innovative way to synthesize novel toxin-neutralizing molecules. It may serve as a strategy for designing a new generation of antivenoms.