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Toxicon 48 (2006) 649–661

www.elsevier.com/locate/toxicon

Role of IgG(T) and IgGa isotypes obtained from arachnidicantivenom to neutralize toxic activities of Loxosceles gaucho,

Phoneutria nigriventer and Tityus serrulatus venoms

Ana Flavia Toroa, Marılia Brinati Maltaa, Sabrina Lucio Soaresa,Guilherme Casoni Da Rochaa, Marcela da Silva Liraa, Thais Abbate De Oliveiraa,Harumi Ando Takeharaa, Monica Lopes-Ferreiraa, Marcelo Larami Santorob,

Rosalvo Guidolinc, Hisako Gondo Higashic, Irene Fernandesa,Katia Cristina Barbaroa,�

aLaboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sao Paulo, SP, BrazilbLaboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sao Paulo, SP, BrazilcPlasma Hyperimmune Section, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sao Paulo, SP, Brazil

Received 14 June 2006; received in revised form 11 July 2006; accepted 13 July 2006

Available online 25 July 2006

Abstract

The ability of IgG(T) and IgGa subclasses—isolated by liquid chromatography from equine arachnidic antivenom

(AAV)—to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms as well as

to remove venom toxins from circulation was investigated. These subclasses showed similar antibody titers against L.

gaucho, P. nigriventer and T. serrulatus venoms, and by immunoblotting few differences were observed in the recognition

pattern of venom antigens. IgG(T) and IgGa neutralized 100% lethality induced by L. gaucho and 50% of P. nigriventer

venom, but IgGa failed to neutralize T. serrulatus venom, in contrast to IgG(T). Both subclasses neutralized local reactions

and dermonecrosis induced by L. gaucho venom in rabbits. In mice, IgG(T) and IgGa partially neutralized the

edematogenic activity induced by P. nigriventer and T. serrulatus venoms, but only IgG(T) neutralized (ca. 81%) the

nociceptive activity induced by T. serrulatus venom. Both subclasses failed to neutralize nociceptive activity induced by P.

nigriventer venom. IgG(T) reduced the serum venom levels of animals injected with L. gaucho, P. nigriventer or T.

serrulatus venoms, while IgGa solely reduced L. gaucho and P. nigriventer venoms levels. Our results demostrate that

IgG(T) and IgGa subclasses neutralize toxic activities induced by P. nigriventer, T. serrulatus and L. gaucho venoms with

different efficacies, as well as depurate these venoms from circulation.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Immunotherapy; Arachnidic antivenom; IgG(T); IgGa; Arachnid; Venom; Loxosceles; Phoneutria; Tityus

front matter r 2006 Elsevier Ltd. All rights reserved

icon.2006.07.029

ng author. Tel.: +5511 37267222x2278/2134;

61505.

sses: kbarbaro@usp.br, , kbarbaro@butantan.

rbaro).

1. Introduction

Loxosceles sp. spiders are distributed in theAmericas, Europe, Australia and Africa, while

.

ARTICLE IN PRESSA.F. Toro et al. / Toxicon 48 (2006) 649–661650

Phoneutria sp. spiders and Tityus sp. scorpions aredistributed in South and Central Americas (White etal., 1995; Hogan et al., 2004). Currently, loxoscelismis considered the most important form of araneismin Brazil due to its high incidence (approximately2000 cases/year), followed by Phoneutria sp. bites(1600 cases/year). Bites by Tityus sp. scorpions aremedically important due to the high number ofnotified accidents (8000 cases/year) and to theirvenom toxicity, especially in children (Ministry ofHealth, 1998).

Usually, the diagnosis is based on patient clinicalpicture at hospital admission. Cutaneous loxosce-lism (84–97% of the accidents) is characterized bydermonecrosis at the bite site. Viscero-cutaneousloxoscelism is observed in more severe cases (3–16%of the patients) and causes, besides the localreaction, intravascular hemolysis, rhabdomyolysisand acute renal failure, the main cause of death insuch accidents (Ministry of Health, 1998; Sezerinoet al., 1998; Franc-a et al., 2002). The use ofimmunotherapy to treat loxoscelism depends onregional experience and its frequency ranges from11.9% to 70.0% in Parana and Sao Paulo States,respectively. Nonetheless, the efficacy of immu-notherapy depends on the interval between thetreatment and the spider bite (Ministry of Health,1998).

The clinical picture of accidents by Phoneutria sp.and Tityus sp. are similar. Approximately 90% ofthe patients present mild symptoms, characterizedmainly by pain and edema at the bite site. Pain istreated with analgesic and/or local anesthesia.Moderate cases present, besides local pain, othermanifestations, e.g., vomiting, psychomotor agita-tion, prostration, sweating, tachycardia and arterialhypertension. Severe cases, practically restricted tochildren, present these symptoms as well as cardiacfailure, pulmonary edema and shock (Ministry ofHealth, 1998). The use of immunotherapy in Tityus

sp. and Phoneutria sp. envenomation depends onthe severity of the clinical symptoms, but its use isvital and indispensable to treat children and adultswith systemic symptoms (Ministry of Health, 1998).

Loxosceles sp. venom is a mixture of proteins andpeptides such as proteases, hydrolases, lipases,hyaluronidases, alkaline phosphatase, 5-ribonucleo-tidase and phosphohydrolases (Futrell, 1992). Thedermonecrotic component of the Loxosceles venom(sphingomyelinase D) seems to be very importantfor triggering pathogenic mechanisms characteristicof Loxosceles envenomation, including complement

activation (Tambourgi et al., 1995), platelet activa-tion (Rees et al., 1988) and endothelial cell-dependent neutrophil activation, which prompt therelease of many cytokines (Malaque et al., 1999;Desai et al., 1999), inflammatory mediators andintercellular adhesion molecules (Patel et al., 1994).Phoneutria sp. and Tityus sp. venoms are a complexmixture of basic proteins of low molecular mass(from 2 to 16 kDa for Phoneutria sp. and o8 kDafor Tityus sp.), which are active on voltage-sensitiveNa+ channel of excitable cells (Cordeiro et al.,1995; Lima and Martin-Eauclaire, 1995). Thesetoxins depolarize the sympathetic and parasympa-thetic nerve terminals facilitating the spontaneousrelease of neurotransmitters, such as acetylcholineand catecholamines (Costa et al., 2001; Teixeira etal., 2001).

Horse IgG isotypes have been designated IgGa,IgGb, IgGc and IgG(T), based on their immunoe-lectrophoretic mobility (Klinman et al., 1965).Ideally, only the effective isotype should be admi-nistered to patients. Arachnidic antivenom (AAV)(by Butantan Institute) is produced by gradualimmunization of horses with repeated sublethaldoses of Phoneutria nigriventer, Tityus serrulatus

and Loxosceles gaucho venoms. In spite of themodernization of antivenom production, they stillcontain a large quantity of non-useful proteins,which contribute to the development of earlyreactions such as itching, rash, malaise as well asanaphylactic shock. In fact, a large range of reactionincidence (14–87%) is observed in different studies(Cupo et al., 1991; Cardoso et al., 1993; Fan et al.,1999).

Taking into consideration that some IgG isotypespresent in AAV might be superfluous, the aim ofthis study was to purify the main IgG isotypes ofserum and to investigate their roles to neutralizesome toxic activities of L. gaucho, P. nigriventer andT. serrulatus venoms, and to determine the venomkinetics in the circulation of mice experimentallyinjected with these isotypes.

2. Materials and methods

2.1. Animals and venoms

Swiss mice (18–20 g) and adult rabbits (3–4 kg)were provided by Butantan Institute Animal House.Specimens of L. gaucho were collected in Sao PauloState. The spiders were kept in quarantine for 1week without food before venom collection. A pool

ARTICLE IN PRESSA.F. Toro et al. / Toxicon 48 (2006) 649–661 651

of venom collected from approximately 1000L. gaucho spiders was used. Venoms of P. nigri-

venter and T. serrulatus were kindly supplied byLaboratory of Arthropods, Butantan Institute. Theprotein content of venom pools was determinedusing bicinchoninic acid (Sigma, USA). The proce-dures involving animals were performed in con-formity with national laws and policies controlledby Butantan Institute Animal Investigation EthicalCommittee (protocol no. 014/2000).

2.2. Antivenoms

AAV was produced by the Butantan Institute, byimmunizing horses with a mixture of venoms of L.

gaucho (21.5%), P. nigriventer (21.5%) and T.

serrulatus (57%) (Cardoso et al., 2003). Previousto pepsin digestion and the manufacturing process,an aliquot was taken out to purify IgGa and IgG(T)isotypes. The commercial arachinic antivenom(cAAV, batch no. 0211124, protein concentration51.3mg/ml) was used as a positive control. Itcontains only F(ab0)2 fragments and is currentlyemployed for the treatment of patients.

2.3. Isolation of horse IgG(T) and IgGa from

antivenom

The IgGa and IgG(T) isotypes were isolated fromAAV by chromatography on protein A-sepharosefollowed by chromatography on a column of anti-horse IgG(T) monoclonal antibody (LO-HoGT-1)-sepharose, as reported by Fernandes et al. (1994).Briefly, a 2.5 cm� 8.0 cm protein A-sepharose col-umn was equilibrated with 0.10M borate-bufferedsaline, pH 8.5 (BBS), 1.5ml of antivenom wasapplied to it, and then the column was washed withBBS until no protein could be detected in theeffluent. The bound IgG isotypes were eluted with0.15M citrate buffer pH 5.5. Isolation of IgG(T)and IgGa isotypes was achieved by passing theeluted mixture in the column of sepharose-LO-HoGT-1 [mouse IgG monoclonal antibody anti-horse IgG(T)]. The first peak, obtained by washingthe column with BBS, contained IgGa, whereas thesecond peak, eluted with 0.15M citrate buffer pH4.0, contained IgG(T). All chromatographic proce-dures were performed at 4 1C. The purity of thesefractions was evaluated by immunoelectrophoresisusing goat anti-horse IgG(T) and rabbit anti-horseIgGa, IgGb and IgGc purchased from BethylLaboratories INC (Montgomery, ALA, USA).

The quantification of IgG isotype content afterpurification was determined as described by Littleand Donahue (1968).

2.4. ELISA

Antibody titers of the different subclasses of AAVwas titrated by ELISA using L. gaucho, P.

nigriventer or T. serrulatus venoms (10 mg/ml) tocoat plates. The reaction was read using an ELISAreader (Multiskan EX) and the titer determined asthe reciprocal of the highest dilution that causes anabsorbance greater than 0.050 at 492 nm, as non-specific reactions were observed below this value.

2.5. SDS– polyacrylamide gel electrophoresis

(SDS– PAGE)

Proteins of L. gaucho, P. nigriventer and T.

serrulatus venoms were analyzed by 12.5%SDS–PAGE under non-reducing conditions. Pro-teins were revealed by Coomassie Blue staining.Myosin (205.73 kDa), b-galactosidase (133.08 kDa),bovine serum albumin (83.92 kDa), carbonic anhy-drase (41.56 kDa), soybean trypsin inhibitor(31.35 kDa), lysozyme (17.26 kDa) and aprotinin(7.02 kDa) were used as molecular mass markers(BioRad, Hercules, CA, USA).

2.6. Western blotting

Proteins of L. gaucho, P. nigriventer and T.

serrulatus (20 mg) were first fractionated bySDS–PAGE as described above and further electro-blotted. Nitrocellulose membranes were incubatedwith AAV (diluted 1/600), IgGa or IgG(T) isotypes(1.0mg/ml, diluted 1:50), and immunoreactiveproteins were detected using peroxidase-labeledanti-horse IgG. The blot was developed with0.05% 4-chloro-1-naphtol in 15% (v/v) methanol,in the presence of 0.03% (v/v) H2O2.

2.7. Lethal dose 50% (LD50)

Mice (n ¼ 6) were observed for 48 h after i.d.venom injection and LD50 was determined by probitanalysis. The LD50’s of L. gaucho, P. nigriventer andT. serrulatus venoms were 0.45, 0.48 and 0.9mg/kg,respectively.

ARTICLE IN PRESSA.F. Toro et al. / Toxicon 48 (2006) 649–661652

2.8. Local reaction and dermonecrotic activity of L.

gaucho venom

The measurement of local reaction (edema/erythemaand paleness/ecchymosis areas) and of dermonecroticactivity was determined by i.d. injection of 5mg ofL. gaucho venom (in 0.2ml of 0.15M NaCl) into therabbit dorsum skin (n ¼ 4). The areas of edema/erythema, paleness/ecchymosis and dermonecrotic wereinspected 48h after injection and reported in cm2.

2.9. Nociceptive and edematogenic activities of T.

serrulatus and P. nigriventer venoms

To detect the nociceptive activity, mice (n ¼ 4)were injected in the right hind paw with 30ml of0.15M NaCl containing different doses of P.

nigriventer and T. serrulatus venoms. Animals wereput individually under glass funnels on a mirror.Afterwards, reactivity of animals was measured, inseconds, taken for animals to lick or bite the injectedpaw, during 30min of experimental evaluation.

Edema-forming activity was evaluated at differ-ent times (5, 15, 30, 120 and 240min) as thedifference of thickness (mm) between the right footpaw injected with different doses of P. nigriventer orT. serrulatus venom samples diluted in 0.15M NaCland the left paw of mice injected with the samevolume of vehicle.

2.10. Neutralization of the local reaction and

dermonecrotic and lethal activities of L. gaucho

venom after pre-incubation with antibodies

The ability of cAAV, IgGa or IgG(T) isotypes(9mg/ml) to neutralize the local reaction anddermonecrotic activity of L. gaucho venom wasestimated by incubating L. gaucho venom (5 mg)with 0.2ml of each antibody sample for 1 h at 37 1C.After incubation, the mixture was centrifuged andthe supernatant injected i.d. into the rabbit dorsum.The local lesion and dermonecrotic areas wereinspected 48 h after injection and were reported incm2. Samples (0.2ml) of cAAV, IgG(T) and IgGaalone were used as negative controls.

2.11. Neutralization of the lethal activity of L.

gaucho, P. nigriventer and T. serrulatus venoms after

pre-incubation with antibodies

In order to determine the ability of the cAAV,IgGa or IgG(T) isotypes (9mg/ml) to neutralize the

lethal activity of venoms, 2 or 3 LD50 of each venomwere mixed with 0.5ml of each antibody sample.The mixture was incubated for 1 h at 37 1C,centrifuged and the supernatant injected i.p. intomice (n ¼ 6). The number of surviving animals wasassessed after 48 h.

2.12. Effective dose 50% (ED50) of IgG(T) and

IgGa isotypes to L. gaucho venom

The neutralizing ability of IgG(T) and IgGaisotypes was expressed as effective dose 50%(ED50), defined as microlitres of antivenom permilligram of venom at which half of the injectedanimals survived. The challenge dose used for L.

gaucho venom was 5 LD50. The ED50 for IgGa andIgG(T) was defined as milligram of protein permilligram of venom. Samples were mixed withvenom dissolved in 0.15M NaCl, and then incu-bated for 1 h at 37 1C. Thereafter, they werecentrifuged to remove the immune complexes andthe supernatants were injected i.p. into mice.Control animals received the same amount ofvenom dissolved in the same volume of salinewithout antibodies. Survival was determined 48 hlater.

2.13. Neutralization of nociceptive and edematogenic

activities of P. nigriventer and T. serrulatus venoms

after pre-incubation with antibodies

The ability of cAAV, IgGa or IgG(T) isotypes(9mg/ml) to neutralize nociceptive activity ofP. nigriventer and T. serrulatus venoms wasestimated by incubating P. nigriventer (2.5 mg) andT. serrulatus (0.5 mg) venoms with 30 ml of eachantibody sample for 1 h at 37 1C. For neutralizationof edema activity, doses of 1.0 and 2.0 mg of P.

nigriventer and T. serrulatus venoms, respectively,were used. After incubation, the mixture wascentrifuged and the supernatant injected into theright hind paw of mice (n ¼ 6), according to theexperimental protocol already described. Resultswere expressed as mean7SEM for each parameterstudied. Samples (30 ml) of cAAv, IgG(T) and IgGaalone were used as negative controls.

2.14. Kinetics of venom removal by cAAV, IgGa or

IgG(T) isotypes in mice sera

Groups of mice (n ¼ 4) were injected i.d. with10 mg of P. nigriventer, T. serrulatus or L. gaucho

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Table 1

Antibody titers of AAV, cAAV, or purified IgGa and IgG(T)

isotypes against L. gaucho, P. nigriventer and T. serrulatus

venoms

Venoms

P. nigriventer T. serrulatus L. gaucho

IgG(T) 64,000 256,000 1,024,000

AAV IgGa 32,000 512,000 1,024,000

IgGb o1000a 4000 16,000

IgGc o1000a 16,000 32,000

Purified IgG(T) 32,000 256,000 1,024,000

Purified IgGa 16,000 256,000 1,024,000

cAAV 512,000 256,000 1,024,000

Antibody titers were determined by ELISA as the reciprocal of

the highest dilution with an absorbance greater than 0.050 at

492 nm, since non-specific reactions were observed below this

value.aValues below initial dilution.

A.F. Toro et al. / Toxicon 48 (2006) 649–661 653

venoms and simultaneously with 0.2ml of normalhorse serum (NHS), cAAV, IgG(T), or IgGaisotypes (9mg/ml) by i.v. route. At different timeintervals after injection (15, 30, 60, 240 and1440min), mice were anesthetized, bled by scissionof axillary plexus and sacrificed. Blood sampleswere placed into 1.5-ml plastic centrifuge tubes,centrifuged at 8000 g for 5min and serum wascollected for venom assay. Venom levels weredetermined by ELISA. Briefly, 96-well microplateswere coated with horse anti-arachnidic serumproduced by Butantan Institute. A standard curve(500–0.7 ng/ml) of each venom was used to deter-mine the amount of venom in the test samples.Rabbit anti-L. gaucho, anti-P. nigriventer or anti-T.

serrulatus venoms sera were used as secondaryantibodies and goat anti-rabbit IgG labeled withperoxidase was used as conjugate. After washing,substrate solution (ortho-phenylenediamine andH2O2) was added to the wells. The color develop-ment was stopped by the addition of 50 ml of 30%(v/v) H2SO4 and the intensity of the color wasmeasured at 492 nm. The lower limit of sensitivity ofthe assay was 2 ng/ml. Results were expressed asmean7SEM for each parameter studied.

2.15. Statistical analysis

Results are presented as means7SEM. Two-wayANOVA followed by Bonferroni test was used toanalyze data (SigmaStat 3.0 software). Values withpo0:05 were considered statistically significant.

3. Results

The IgGa and IgG(T) isotypes were isolated fromAAV by a combination of two affinity chromato-graphic procedures, protein A-sepharose followedby anti-IgG(T) monoclonal antibody (LO-HoGT-1)-sepharose. The purity of these fractionatedisotypes was confirmed by immunoelectrophoresis(data not shown). Around 850mg of IgG(T) and980mg of IgGa were recovered from 100ml ofAAV.

Table 1 shows the titers of IgGa, IgGb, IgGc andIgG(T) subclasses present in AAV for each venomstudied. Similar results were verified for IgG(T) andIgGa isotypes for each venom, since only differencesabove two dilution steps were considered signifi-cant. Interestingly, higher antibody titers weredetected against L. gaucho venom. Lower levels ofIgG(T) and IgGa against P. nigriventer venom were

detected. Low IgGb and IgGc subclass titers weredetected in AAV, and the highest ones were alsoobserved against L. gaucho venom. Similar titers ofIgG(T) and IgGa purified from AAV were observedfor each venom. This result showed that purifiedIgG(T) and IgGa maintain the same proportionfound in AAV, indicating that almost no loss ofantibodies occurred during the purification process.On the other hand, cAAV (commercial antivenom)showed a titer range varying from 256,000 to T.

serrulatus, 512,000 to P. nigriventer venom and upto 1,024,000 to L. gaucho venom.

The electrophoretic profile (Fig. 1) shows that L.

gaucho venom presented a major band of 35 kDaand other components were located between 22 and203 kDa. In T. serrulatus venom, bands weredistributed along 8.4–75 kDa, while in P. nigriventer

venom components were located between 7.9 and135 kDa. Many components below 15 kDa werepresent in all 3 venoms. As shown in Fig. 1, bothIgG(T) and IgGa showed similar patterns ofrecognition to AAV. Different intensities of colora-tion of some bands were observed mainly forP. nigriventer venom, whose components above33 kDa were weakly recognized by AAV, IgGa andIgG(T) isotypes. Components below 28 kDa of P.

nigriventer venom were not recognized by bothisotypes as well by AAV.

Once antigens could be recognized by isolatedisotypes, we investigated the capacity of antivenomand their isotypes to neutralize some toxic activitiesof L. gaucho, P. nigriventer, and T. serrulatus

venoms.

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Fig. 1. Electrophoretic profile of L. gaucho (Lg), T. serrulatus (Ts) and P. nigriventer (Pn) venoms obtained by 12.5% SDS–PAGE (left).

Components of L. gaucho (Lg), T. serrulatus (Ts) and P. nigriventer (Pn) venoms were fractionated by SDS–PAGE and revealed by

Western blotting (right) using arachnidic antivenom serum (AAV), or purified IgGa or IgG(T) isotypes. The numbers at right indicate the

position of molecular mass markers.

Table 2

Neutralization of lethal activity of L. gaucho, P. nigriventer and T. serrulatus venoms by cAAV, IgGa and IgG(T)

P. nigriventer T. serrulatus L. gaucho

48 h %a 48h % 48h %

Survival (2 DL50)

Venom+PBS 0/6b 0 2/6 33.3 1/6 16.6

Venom+cAAV 6/6 100 6/6 100 6/6 100

Venom+purified IgG(T) 3/6 50 6/6 100 6/6 100

Venom+purified IgGa 3/6 50 2/6 33.3 6/6 100

Survival (3 DL50)

Venom+PBS 0/6b 0 1/6 16.7 0/6 0

Venom+cAAV 6/6 100 6/6 100 6/6 100

Venom+purified IgG(T) 1/6 16.7 1/6 16.7 6/6 100

Venom+purified IgGa 1/6 16.7 1/6 16.7 3/6 50

aPercentual of neutralization of lethal activity 48 h after venom injections. L. gaucho, P. nigriventer or T. serrulatus venom (2 or 3 LD50)

was incubated with 0.5ml of commercial arachnidic antivenom (cAAV) or with their IgGa or IgG(T) isotypes (9.0mg/ml) for 1 h at 37 1C.

The sample was centrifuged and the supernatant injected i.p. in mice.bLive/injected animals.

A.F. Toro et al. / Toxicon 48 (2006) 649–661654

As shown in Table 2, both purified isotypesprotected animals of the lethality induced by 2 or 3LD50 of L. gaucho venom. Smaller amounts ofIgG(T) (ED50: 6.1mg of IgG(T)/mg venom—95%confidence limit, 2.9–12.7mg) than IgGa (ED50:15.2mg of IgGa/mg venom—95% confidence limit,9.4–24.6mg) were necessary to neutralize thelethality induced by 5 DL50 of L. gaucho venom.However, IgGa protected only 50% of mice injected

with 3 DL50 of L. gaucho venom. On the otherhand, IgG(T) protected against the lethality inducedby 2 LD50 of T. serrulatus (100% mice) and P.

nigriventer (50% mice) venoms. Whereas, IgGaprotected 50% mice of lethality induced by 2 LD50

of P. nigriventer venom, but failed to protect theanimals injected with 2 LD50 T. serrulatus venom.

Both isotypes were efficacious to neutralizedermonecrosis (Fig. 2A) and local reaction

ARTICLE IN PRESSA.F. Toro et al. / Toxicon 48 (2006) 649–661 655

(Fig. 2B and C) caused by L. gaucho venom.However, IgGa was less potent than IgG(T), sincesmaller amounts (0.9mg/ml) of this antibody werenot as effective in neutralizing dermonecrosis (datanot shown).

Nociceptive activity induced by P. nigriventer

venom (2.5 mg) was not neutralized by any purifiedisotypes (Fig. 3A). However, IgG(T) was efficacious(po0:001) to decrease nociceptive activity inducedby T. serrulatus venom (0.5 mg) (Fig. 3B). OnlycAAV was able to neutralize the nociceptive activityinduced by both venoms.

Both IgG(T) and IgGa isotypes reduced in about50–67%, respectively, the edema in the first 30minafter P. nigriventer venom injection (Fig. 4A).Neutralization of edema (67–87%) occurred when

Fig. 2. Areas of dermonecrosis (A), edema/erythema (B), ecchymosis/p

0.2ml of PBS (Ven), cAAV, IgG(T) or IgGa (9mg/ml) for 1 h at 37 1C a

were measured 48 h after venom injection. Results are expressed as me

with venom alone.

T. serrulatus venom was incubated with bothisotypes in the first 30min (Fig. 4B). Besides, asignificant decrease in edema was also observed120min after T. serrulatus venom injection for IgGaand IgG(T) (76–86%, respectively). Only at 5minafter injection of T. serrulatus venom plus cAAV,the edema reduction was statistically significantwith animals injected only with venom (control). Noneutralization by cAAV was observed in P.

nigriventer venom. Unspecific edema was observedwhen cAAV was injected alone in almost all timeperiods analyzed.

In order to determine the kinetics of venom incirculation after injection of cAAv and isotypes,blood was collected at different time periods afterinoculation, and assayed for venom by ELISA.

aleness (C) areas induced by 5mg of L. gaucho pre-incubated with

nd injected i.d. into the rabbit dorsum (n ¼ 4). The affected areas

an7SEM. *Statistical difference (po0:05) with animals injected

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Fig. 3. Neutralization of nociceptive activity. P. nigriventer

(2.5mg, A) or T. serrulatus (0.5mg, B) venom was incubated with

30ml of PBS (Ven), AAV, cAAV, IgGa or IgG(T) (9mg/ml) for

1 h at 37 1C and injected in the right foot paw of mice.

Nociception was estimated as the time in seconds spent by

animals licking the envenomed paw during a 30min observation

time. Negative controls were injected with cAAV, IgGa or IgG(T)

alone. *Statistical difference (po0:05) between the groups

injected with antibodies and venom alone.

A.F. Toro et al. / Toxicon 48 (2006) 649–661656

Venom levels peaked in circulation within the firsthour after venom injection. When antibodies wereadministered concomitantly, both cAAV andIgG(T) were efficacious in removing P. nigriventer

(Fig. 5A) and T. serrulatus (Fig. 5B) venoms fromcirculation, as well as protecting against the lethalactivity induced by them. IgGa isotype was lesseffective in removing P. nigriventer venom fromcirculation when compared with IgG(T) and cAAV,and it failed to remove T. serrulatus venom withinthe first hour after injection. IgGa was notefficacious in protecting the lethal activity causedby P. nigriventer and T. serrulatus venoms, since

some animals died between 4 and 24 h afterinjection. Both isotypes and cAAV completelyremoved L. gaucho venom from circulation at alltime periods tested (Fig. 5C). No venom was foundin circulation 24 h after injection, for all threevenoms tested. Control animals injected only withvenoms died before 24 h.

4. Discussion

Since immunotherapy was introduced in Brazil acentury ago, several modifications and improve-ments in purification processes have been accom-plished, but many questions are still not completelyresolved, especially those concerning the effectiveability of antibodies to neutralize venom toxins.Investigations dealing with this issue can promoteimprovements in treatment efficacy. The aim of thisproject was to verify the role of each isotype in theprotection induced by AAV used for treatment ofPhoneutria sp., Loxosceles sp. and Tityus sp. bites inBrazil. These venomous animals are responsible for34% of all bites reported to Ministry of Health(1998).

Both IgG(T) and IgGa isotypes are majorcomponents of AAV, and the latter is present inan amount 15% higher than IgG(T). These dataagree with those obtained by McDougall (1975)demonstrating that IgGa and IgG(T) accounts for80% of all immunoglobulins present in normalequine serum. By ELISA, we noted that few lossesof immunoglobulin isotypes occurred during thepurification process (data not shown). Smallamounts of IgGb and IgGc were detected. Thus,we can speculate that these isotypes do not have asignificant neutralizing effect.

No important differences between IgG(T) andIgGa were detected by ELISA and immunoblottingfor each venom studied. However, we detectedhigher antibody titers against L. gaucho venom andlower ones to P. nigriventer venom, but nosignificant difference was observed for cAAV anti-body titers for all three venoms tested.

The proportion of P. nigriventer venom antigen inthe venom pool (21.5%) used for immunization ofhorses can be one of the factors responsible forlower antibody titers, since this venom is mainlycomposed of peptides and, therefore, less immuno-genic. On the other hand, Loxosceles sp. venomcomponents are very immunogenic and inducehigher antibody titers (Barbaro et al., 1994, 1996a).

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Fig. 4. Neutralization of edematogenic activity. P. nigriventer (1.0mg, A) or T. serrulatus (2.0mg, B) venom (Ven) was incubated with 30 mlcAAV, IgGa and IgG(T) (9mg/ml) for 1 h at 37 1C and injected in the right foot paw of mice. Edema-forming activity was evaluated as the

difference of thickness (mm) between the right foot paw, which received the test sample, and the left paw, injected with the same volume of

vehicle, at different time periods. Negative controls were injected with cAAV, IgGa or IgG(T) alone. *Statistical difference (po0:05) withanimals injected with venom alone.

A.F. Toro et al. / Toxicon 48 (2006) 649–661 657

The electrophoretic profile of Loxosceles gaucho

venom presented a major band of 35 kDa, which isresponsible for its dermonecrotic and lethal activity(Barbaro et al., 1996b). This component was

remarkably recognized by IgG(T) and IgGa byWestern blotting. Many immunogenic componentsbetween 28 and 205 kDa of T. serrulatus andP. nigriventer venoms were detected by cAAV,

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Fig. 5. Detection of P. nigriventer (A), T. serrulatus (B) or L. gaucho (C) venoms in serum after intradermal injections. Groups of mice

(n ¼ 4) injected i.d. with 10 mg of each venom (Ven) were simultaneously administered with 0.2ml of normal horse serum (NHS), cAAV, or

purified IgG(T) or IgGa isotypes (9mg/ml). At different time periods after injection (15, 30, 60, 240 and 1440min), mice were bled and sera

collected for assay of venom. *Statistical difference (po0:05) with animals injected with venom plus normal horse serum (NHS). an ¼ 1;#n ¼ 2; bn ¼ 3. gSamples not assayed because animals died after 240min.

A.F. Toro et al. / Toxicon 48 (2006) 649–661658

IgG(T) and IgGa. Our electhrophoretic and Wes-tern blotting data are in agreement with thosereported by Barbaro et al. (1996b), who also verifiedsimilar profiles of recognition for P. nigriventer, T.

bahiensis and L. gaucho venoms by cAAV. How-ever, using our protocol, it was not possible to verifyif there are antibodies that recognize the neurotoxicpeptides below 7 kDa, which are present in T.

serrulatus and P. nigriventer venoms, as describedby Lima and Martin-Eauclaire (1995).

Our results show that IgG(T) was more effica-cious than IgGa in protecting against the lethalityinduced by T. serrulatus and L. gaucho venoms.Both isotypes have less ability to protect animalsagainst the lethal activity of P. nigriventer venom.

Both isotypes failed to protect animals injected withP. nigriventer and T serrulatus venoms using highervenom doses (3 LD50). IgG(T) neutralized comple-tely only the activity of L. gaucho venom (3 LD50),while IgGa protected 50% of animals. These resultswere confirmed by the values of ED50, whichdemonstrated that 2.5 times less IgG(T) than IgGais necessary to neutralize the lethality induced by L.

gaucho venom (5 LD50).Under our experimental conditions, the dermo-

necrotic activity induced by L. gaucho venom wascompletely neutralized by both isotypes. Similarresults were observed for edema/erythema andecchymosis/paleness areas. However, IgG(T) wasmore efficacious than IgGa when smaller amounts

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of these antibodies were used (data not shown). Theability of specific antibodies to neutralize the in vivodermonecrotic activity of Loxosceles venom hadalready been described by many authors (Barbaro etal., 1994, 2005; Gomez et al., 1999). Guilherme et al.(2001) mimicked the treatment employed in patientsbitten by Loxosceles sp. spiders by administratingantivenoms i.v. in rabbits, confirming the impor-tance of antibodies for neutralization of dermone-crosis, even when administrated some hours afterenvenomation.

Both cAAV and IgG(T) isotypes neutralizednociceptive activity induced by T. serrulatus venom.However, no isotypes neutralized nociceptive activ-ity induced by P. nigriventer venom. Probably,P. nigriventer venom can activate other endogenoussystems (Marangoni et al., 1993; Bento et al., 1995;Zanchet et al., 2004). Another explanation is thatthe amount of isotypes used in our protocol wasinsufficient to neutralize this venom, since AAV wasable to neutralize this activity.

We observed that IgG(T) and IgGa reduced about50% of edema activity. On the other hand, cAAValone induced edema, which was exacerbated byvenom inoculation. Possibly, preservatives used inmanufacturating cAAV, e.g., formaldehydes, caninduce edema by themselves, promoting a rapidrelease of endogenous inflammatory mediators, whichcould then be potentialized by venom injection.

It is interesting to observe the contrast betweenthe neutralization of nociceptive and edematogenicactivities elicited by purified isotypes. While edemainduced by either P. nigriventer or T. serrulatus

venoms was partially inhibited by both isotypes,only IgG(T) neutralized the nociceptive activityinduced by T. serrulatus venom. Taking these datatogether, we can speculate that different mechan-isms are responsible for nociceptive and edemaactivities, which are independent phenomena. Onthe other hand, such isotypes ameliorated thegeneral conditions of systemically envenomed ani-mals (data not shown). This result indicated thatantibodies are indeed efficacious in neutralizing thesystemic effects induced by P. nigriventer and T.

serrulatus venoms.The ability of isotypes to neutralize more

efficaciously the lethal activity of L. gaucho venomthan that of T. serrulatus and P. nigriventer venomsis probably due to the higher immunogenicity of L.

gaucho venom (Barbaro et al., 1994), which cangenerate higher amounts of neutralizing antibodies.We can also speculate that peptides present in

P. nigriventer and T. serrulatus venoms are respon-sible for envenomation symptoms, but they cannotinduce an effective immune humoral response dueto their low molecular mass. In addition, theamount of venom used for the hyper-immunizationof horses could also influence the production ofneutralizing antibodies. Certainly, the amount ofL. gaucho venom used for the immunization ofhorses was sufficient to produce specific antibodiesagainst the toxic components of venom. On theother hand, the increased efficiency of the IgG(T)isotype in neutralizing T. serrulatus venom, com-pared to P. nigriventer venom, can be ascribed to theamount of T. serrulatus venom used in theimmunization of horses, which is more than twofoldhigher than that of P. nigriventer venom. This factwas also confirmed for the lower antibody titersagainst P. nigriventer present in AAV as well as inboth purified isotypes. Besides, antibody concentra-tion is a fundamental factor for evaluating theirneutralizing capacity, since our findings showed thatcAAV, but not the purified isotypes, completelyneutralized the lethal activity of P. nigriventer andT. serrulatus venoms. In fact, while cAAV contained51.3mg/ml of total proteins, the purified isotypeswere used at 9mg/ml, and such difference inantibody concentration could explain the lowerefficiency presented by the purified isotypes whencompared to cAAV.

We also investigated the efficiency of antibodiesto remove venom from murine circulation. Highlevels of venoms were already detected within thefirst 15min after injection. After 4 h, a pronouncedreduction in venoms levels occurred, corroboratingresults described in literature for P. nigriventer

(Chavez-Olortegui et al., 2001), T. serrulatus (San-tana et al. 1996) and T. discrepans (Sevcik et al.,2004) venoms. cAAV, IgG(T) or IgGa injectedsimultaneously with each venom could totallyremove them from circulation. CAAV, whencompared with purified isotypes, more rapidlyremoved T. serrulatus and P. nigriventer venomsfrom circulation. These data agree with those ofRevelo et al. (1996), who observed a rapid reductionof T. serrulatus venom in mice serum after admin-istration of scorpionic antivenom. However, weobserved a residual level of T. serrulatus and P.

nigriventer venoms in sera of animals injected withIgG(T) and IgGa. IgG(T) was more effective thanIgGa in the removal of T. serrulatus venom fromcirculation within the first 60min but at hour four,both subclasses were similarly effective in venom

ARTICLE IN PRESSA.F. Toro et al. / Toxicon 48 (2006) 649–661660

removal. For P. nigriventer venom, both subclasseswere equally efficacious on venom removal. Signsand symptoms of envenomation, such as sweating,hyperspitting, lacrimation, and agitation—werereduced in animals, which received either cAAV orpurified isotypes, confirming the importance ofantibodies to neutralize the symptoms of systemicenvenomation. In our experiments we noticed thatIgG(T) was more effective than IgGa in neutralizingthe most toxic activities of T. serrulatus and L.

gaucho venoms. Both IgG(T) and IgGa were similarto neutralize P. nigriventer venom. These results arein ability to agreement with previous data reportedby us (Fernandes et al., 1997, 2000a, b). IgG(T)isolated from anti-bothropic or anti-crotalic serafrom Butantan Institute, Brazil, was about three-fold and seven-fold more protective than IgGa forneutralization of lethal activity of Bothrops jararaca

and Crotalus durissus terrificus venoms, respectively.Interestingly, in the antivenom produced by Clodo-miro Picado Institute, Costa Rica, neutralizingantibodies were almost exclusively of IgG(T)isotype. Therefore, IgG(T) is the main IgG subclasspresent in equine hyperimmune sera produced byButantan and Clodomiro Picado Institutes, and isalso the main isotype responsible for neutralizationof hemorrhagic, coagulant and phospholipase A2

(indirect hemolytic) activities, followed by IgGa.Thus, the results shown here provide evidence of

a higher neutralizing efficiency of the IgG(T)isotype. However, we cannot conclude that therewas a polarization of the immune response in favorof the production of IgG(T), since high amounts ofIgGa with neutralizing activity were also produced.Besides, we cannot rule out the hypothesis that forsome toxic activities—such as the edema caused byT. serrulatus or P. nigriventer venoms, or the lethalactivity induced by P. nigriventer venom–a syner-gistic action between IgGa and IgG(T) might benecessary for neutralizing toxic activities, since bothof them can partially neutralize such venom effects.On this basis, we would suggest to manufacturers tointroduce chromatographic steps in their produc-tion protocols, at least for L. gaucho and T.

serrulatus venoms, aiming for the purification ofIgG(T), so that a consequent reduction in totalprotein concentration of antivenoms and mainte-nance of high neutralizing titers could be achieved.

It is known that immunotherapy can causeadverse reactions in patients due to the administra-tion of heterologous proteins. One of the factorsthat determine the occurrence of such reactions is

the purity of antivenoms (Ministry of Health, 1998).Thus, the use of purified isotypes could be a newand important step to improve immunotherapy,since the amount of non-useful proteins given topatients, is certainly one of the major causes ofundesirable side effects, which could be reduced.

Acknowledgments

This work was supported by FAPESP (00/00113-0). A.F. Toro was a CNPq fellowship. The authorsthank Nancy F. Kodera and Letıcia M.P. Martinsfor technical assistance.

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