Short communication

Anticomplementary activity of equine whole IgG antivenoms:

comparison of three fractionation protocols

Guillermo Leon, Bruno Lomonte, Jose Marıa Gutierrez*

Instituto Clodomiro Picado, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Rica

Received 5 May 2004; accepted 26 July 2004

www.elsevier.com/locate/toxicon

Abstract

Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with

anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid

fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid

fractionation followed by b-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange

chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemical

characteristics and anti-Bothrops asper venom antibody titers, except that ion-exchange purified antivenom had a lower protein

concentration. Antivenoms fractionated by using b-propiolactone or filtration through quaternary ammonium membrane had a

significantly reduced in vitro ACA. A preparation of caprylic acid-fractionated antivenom was heated in order to induce the

formation of protein aggregates; however, its ACA was similar to non-heated antivenom. None of the antivenoms affected

the hemolytic activity of serum complement in rabbits after a bolus intravenous administration. It is concluded that

(a) b-propiolactone and quaternary ammonium membranes significantly reduce in vitro ACA of caprylic acid-fractionated

equine antivenom, and (b) the validity of in vitro ACA as a predictor of EAR needs to be reexamined in clinical and

experimental studies, since it may not adequately predict in vivo complement activation by antivenoms.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: Antivenom; Anticomplementary activity; Early adverse reactions; IgG; CH50

Parenteral administration of horse or sheep-derived

antivenoms constitutes the only scientifically validated

treatment for snakebite envenomation worldwide

(Theakston et al., 2003; Lalloo and Theakston, 2003).

Despite their well demonstrated clinical efficacy, adminis-

tration of antivenoms is often associated with the appear-

ance of early adverse reactions (EARs), with a highly

variable incidence reported for different products (Cardoso

et al., 1993; Chippaux et al., 1998; Dart and McNally, 2001;

Lalloo and Theakston, 2003; Gawarammana et al., 2004).

The severity of EARs ranges from mild manifestations, i.e.

urticaria, to more complicated reactions involving bronch-

ospasm and hypotension (Lalloo and Theakston, 2003).

0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2004.07.025

* Corresponding author.

The mechanisms involved in EAR are not completely clear

at present. It has been proposed that de novo complement

activation, or anticomplementary activity (ACA), is an

important mechanism associated with EARs (Sutherland,

1977; Lalloo and Theakston, 2003), and this mechanism has

been also implicated in some adverse reactions occurring

after administration of human immunoglobulin preparations

(Barandun et al., 1962). On this basis, it has been assumed

that pepsin digestion of horse antivenom IgG, with the

consequent removal of Fc fragment, would reduce the

ACA of antivenoms (see for example Grandgeorge et al.,

1996). Nevertheless, F(ab 0)2 antivenoms still present ACA

(Sutherland, 1977; Morais et al., 1994; Leon et al., 2001).

Moreover, clinical studies carried out with many antive-

noms have demonstrated a conspicuous variability in terms

of incidence of EAR; in some cases, pepsin-digested F(ab 0)2

Toxicon 45 (2005) 123–128

G. Leon et al. / Toxicon 45 (2005) 123–128124

antivenoms have induced EAR in more than 75% of patients

(Moran et al., 1998; Gawarammana et al., 2004), whereas

other F(ab 0)2 products induce EAR in less than 10% of

patients (Chippaux et al., 1998). Moreover, whole IgG

antivenoms prepared by caprylic acid fractionation of horse

plasma have been shown to induce a low incidence of EAR

(Otero-Patino et al., 1998; Otero et al., 1999). Therefore, it

seems that the physicochemical characteristics of antive-

noms, such as presence of non-IgG proteins and aggregates,

may play a more relevant role in the incidence of EAR than

the presence of Fc fragment in intact IgGs. Protein

aggregates and non-IgG proteins may thus be involved in

the ACA of antivenoms.

Fractionation of horse plasma with caprylic acid is a

simple and convenient method for antivenom production

which has been adapted for industrial production (Rojas

et al., 1994). The clinical performance of various whole IgG

antivenoms obtained with this methodology has been highly

satisfactory in terms of efficacy and safety (Otero-Patino

et al., 1998; Otero et al., 1999). However, such antivenoms

still present ACA when tested on human complement in

vitro (Otero et al., 1999; Leon et al., 2001). Owing to the

potential relevance of ACA in the development of EAR

associated with antivenom administration, in this work we

compared three different fractionation protocols, all of them

involving caprylic acid precipitation of non-IgG proteins, in

order to find novel ways to reduce ACA.

The same batch of hyperimmune horse plasma was used

throughout this study. It was obtained from a group of 30

horses immunized with a mixture of equal parts of the

venoms of Bothrops asper, Crotalus durissus durissus and

Lachesis stenophrys (Angulo et al., 1997). Plasma was

fractionated by three different protocols: (1) Caprylic acid

precipitation of non-IgG proteins, as routinely performed at

the Production Division of Instituto Clodomiro Picado

(Rojas et al., 1994). Briefly, caprylic acid was added to horse

plasma, at a ratio of 6 ml acid per 100 ml plasma, and the

mixture was stirred for 1 h at 20–22 8C. The mixture was

then filtered through a 8 mm paper filter, and desalted and

concentrated by diafiltration. Afterwards, the preparation

was formulated in order to have 0.15 M NaCl, 2.5 g/l phenol

and 0.05 g/l thimerosal, pH 7.0, Finally, the antivenom was

sterilized by filtration through 0.22 mm membrane and

bottled in 10 ml glass flasks. (2) Five millilitres of a solution

of 2 g/l b-propiolactone were added to 95 ml of antivenom

fractionated as in protocol (1). The mixture was stirred for

2 h at 20–22 8C and then allowed to stand overnight at 4 8C.

Afterwards, the preparation was extensively dialyzed

against 0.15 M NaCl, 2.5 g/l phenol and 0.05 g/l thimerosal,

pH 7.0 and the preparation sterilized as described.

(3) Aliquots of 10 ml of antivenom purified by protocol

(1) were filtered through a strongly basic quaternary

ammonium cellulose microporous membrane (Sartobind

Q-15 membrane, Sartorius, Germany). After filtration of

each 10 ml aliquot, the membrane was regenerated by

passing 10 ml of 1N NaOH and 10 ml of sterile 0.15 M

NaCl solution. A total of 100 ml of antivenom were filtered.

At the end, the preparation was sterilized as described.

Antivenoms resultant from the three fractionation protocols

were formulated to have 0.15 M NaCl, 2.5 g/l phenol and

0.05 g/l thimerosal, with a pH of 7.0. In addition, a sample

with an increased content of protein aggregates was

prepared by heating 200 ml of antivenom fractionated as

in protocol (1) during 90 min at 60 8C in a water bath.

b-propiolactone and ion-exchange steps were selected

because these procedures have been proposed to improve

purity and safety of human IgG preparations (Stephan,

1975) and equine antivenoms (Guidolin et al., 1997;

Grandgeorge et al., 1996; Jones and Landon, 2003).

The following parameters were comparatively analyzed:

(1) Total protein concentration was determined by the

Biuret method (Schosinsky et al., 1983). (2) Turbidity was

evaluated by determining the absorbance of undiluted

antivenoms at 590 nm (Rojas et al., 1994). (3) Aggregate

content was analyzed by analytical gel filtration FPLC in a

Superdex 200 column (HR 10/30) using phosphate-

buffered saline solution (PBS) as eluent (Garcıa et al.,

2002). (4) Electrophoresis was carried out on cellulose

polyacetate membranes (Sephrapore III, Gelman Instru-

ments, Michigan) in barbital buffer, pH 8.6, at 180 V.

Strips were stained with Amidoblack and destained with

0.9N acetic acid. (5) Antibody titers against B. asper

venom were determined by ELISA. Ten micrograms

venom, dissolved in 100 ml PBS were adsorbed onto

Immulon-2 microplates (Dynatech, Virginia). After wash-

ing, the wells were blocked with PBS-2% bovine serum

albumin, and various dilutions of the antivenoms were

added and incubated for 1 h. After washing, a peroxidase-

conjugated anti-equine IgG was added. After a final

washing step, substrate (o-phenylenediamine and H2O2)

were added and the reaction stopped with 2 M HCl, before

recording the absorbances at 492 nm in a Dynatech

microplate reader. In all these determinations, experiments

were run in triplicates.

In vitro ACA on human complement was assessed as

follows: Briefly, 2 ml of various dilutions of antivenoms

were added to 0.5 ml of fresh human serum previously

diluted 1:20 with barbital buffer, pH 7.6. After 1 h of

incubation at 37 8C, 100 ml of sheep erythrocytes, pre-

viously sensitized with rabbit anti-sheep erythrocyte

antibodies, were added, and the mixture was incubated 1 h

at 37 8C. Then, 2 ml of barbital buffer were added, the

samples centrifuged and the absorbances of the supernatants

were recorded at 540 nm. ACA activity was expressed as the

inverse of the dilution that reduced complement activity by

50%, taking as 100% the complement activity of serum

samples that were incubated with barbital buffer instead of

antivenom. In vivo ACA was assessed in groups of 10

rabbits (2.5–3.0 kg) injected intravenously (i.v.) with a

bolus of 10 ml antivenom. One hour after injection, rabbits

were bled and the hemolytic activity of complement (CH50)

was determined (Montero et al., 1989). This time interval

Table 1

Protein concentration, content of aggregates, turbidity, and antibody titer of the four antivenoms compared in this studya

Antivenom Protein (g/dl) Aggregates (%)b Turbidity (A590)c Antibody titer (A492)d

Caprylic 4.18G0.13 2.8G0.4 0.045G0.002a 1.408G0.043

CaprylicC b-propiolactone 3.88G0.12 3.2G0.5 0.021G0.003b 1.343G0.058

CaprylicCion-exchange 3.70G0.11* 2.2G0.3 0.020G0.001b 1.580G0.104

Caprylic heated 4.13G0.16 6.8G0.6** 0.072G0.006c 1.502G0.195

*Significantly lower (P!0.05) than protein concentration in antivenoms fractionated with caprylic acid only, both heated and non-heated.

**Significantly higher (P!0.05) than values of the rest of antivenoms.a Results are presented as meanGSD (nZ3).b Percentage of antivenom protein corresponding to high molecular mass aggregates, as judged by FPLC gel filtration.c Turbidity was assessed by determining the absorbance at 590 nm of undiluted antivenom samples. Values with different superscripts (a,b,c)

are significantly different between them.d Tested against B. asper venom. Results correspond to the absorbance at 492 nm in samples in which antivenom was diluted 1:9000.

G. Leon et al. / Toxicon 45 (2005) 123–128 125

was selected on the basis of results obtained in a previous

study (Montero et al., 1989). For each rabbit, the CH50 was

determined in parallel in a sample collected immediately

before antivenom infusion. Complement hemolytic activity

was expressed as the percentage change of CH50 of samples

collected after antivenom administration, as compared with

preinjection samples. All the results are presented as

meanGSD, and the significance of the differences between

Fig. 1. Anticomplementary activity (ACA) of antivenoms in vitro (A) and

human serum and incubated. Then, sheep erythrocytes sensitized with rab

and centrifuged, and the extent of hemolysis assessed by recording the abso

samples incubated with buffer instead of antivenom represent 100% compl

that reduced hemolytic activity of complement by 50%. Assays were

intravenously into groups of 10 rabbits (2.5–3.0 kg). One hour later, rabbit

vivo complement hemolytic activity. For each rabbit, CH50 was also deter

Hemolytic activity of serum was expressed as percentage, taking as 100%

before antivenom infusion. Results are presented as meanGSD. Identificat

antivenom fractionated with caprylic acid followed by b-propiolactone tr

filtration in an ion-exchange membrane of quaternary ammonium; and (4) a

when compared with groups (1) and (4).

mean values of experimental groups was determined by

ANOVA, followed by Tukey–Kramer test.

As shown in Table 1, the four antivenoms had a similar

protein concentration, although antivenom passed through

the ion-exchange membrane had a significantly reduced

protein content when compared with that of caprylic acid

heated and non-heated antivenoms. Heated antivenom

presented a larger content of aggregates, and showed

in vivo (B). (A) Various dilutions of each antivenom were added to

bit anti-erythrocyte antibodies were added. Samples were incubated

rbances of the supernatants at 540 nm. Values obtained from serum

ement activity. ACA was expressed as the reciprocal of the dilution

performed in triplicate. (B) Antivenoms (10 ml) were injected

s were bled and the CH50 of serum was determined as an index of in

mined in serum samples collected before antivenom administration.

the value of CH50 of samples of each individual rabbit collected

ion of antivenoms: (1) antivenom fractionated with caprylic acid; (2)

eatment; (3) antivenom fractionated with caprylic acid followed by

ntivenom fractionated with caprylic acid and then heated. * P!0.05

G. Leon et al. / Toxicon 45 (2005) 123–128126

a significantly higher turbidity than the other antivenoms.

There were no significant differences between antivenoms

in the antibody titer against the venom of B. asper (Table 1).

No overt differences were observed in the electrophoretic

patterns of antivenoms in polyacetate membranes, charac-

terized by a conspicuous band in the gamma- and beta-

glogulin regions (results not shown). The four antivenoms

showed ACA on human complement in vitro (Fig. 1A).

b-Propiolactone and ion-exchange-fractionated antivenoms

had a significantly lower ACA activity than antivenoms

fractionated by caprylic acid alone (Fig. 1A); b-propiolac-

tone-treated antivenom presented the lowest ACA. In

contrast, when antivenoms were administered i.v. to rabbits,

the hemolytic activity of complement was not significantly

reduced, even in the case of heated antivenom (Fig. 1B).

Our results demonstrate that addition of b-propiolactone

or purification through an ion-exchange membrane signifi-

cantly reduces the ACA of equine whole IgG antivenom

which was initially fractionated by caprylic acid precipi-

tation of non-IgG proteins. b-propiolactone has been

previously shown to reduce ACA of human IgG prep-

arations (Stephan, 1975) and anaphylaxia induced by equine

antivenoms and antitoxins in guinea pigs (Guidolin et al.,

1997), as well as to inactivate virus in the manufacture of

human immunoglobulin preparations for intravenous use

(Burnouf and Radosevich, 2000). On the other hand, ion-

exchange chromatography on anion exchangers has been

widely used for human IgG purification (Jungi et al., 1986;

Tanaka et al., 2000) and in antivenom production, as

a purification step after initial antibody fractionation

(Grandgeorge et al., 1996; Saetang et al., 1997; Jones and

Landon, 2003). Thus, it is suggested that these methods

could be included in antivenom fractionation protocols after

IgG purification by caprylic acid precipitation to further

purify whole IgG antivenoms and to reduce their ACA. The

characteristics of the antivenoms resulting from the three

protocols tested are highly similar in terms of aggregate

content, electrophoretic pattern and antibody titer, although

the use of ion-exchange membrane filtration caused a small

reduction in protein concentration, probably due to the

removal of traces of non-IgG serum proteins. A somehow

surprising finding was that heated antivenom, which

presented a higher content of aggregates, showed a similar

ACA in vitro that antivenom fractionated with caprylic acid

only. It is likely that, in order to increase ACA, a higher

content of aggregates is required. A stronger heat treatment

is probably required to achieve such higher aggregate

antivenom.

Despite the observation of ACA of antivenoms in vitro,

no such activity was detected in rabbits in vivo after a bolus

i.v. administration. These observations raise doubts on the

clinical significance of the in vitro ACA test as a predictor of

ACA in vivo. Various workers have described ACA activity

by antivenoms in vitro (Sutherland, 1977; Montero et al.,

1989; Otero et al., 1999; Leon et al., 2001), but evidence of

ACA in vivo in snakebitten patients receiving antivenoms is

weak. One of the most detailed studies performed on this

subject did not demonstrate complement activation in vivo

in a number of patients receiving various antivenoms,

despite the observation of ACA activity of these products in

vitro (Malasit et al., 1986). In our case, the dose

administered in rabbits (3–4 ml/kg) is higher than the dose

usually administered in humans, which roughly corresponds

to 1–2 ml/kg (Gutierrez, 1995). It may be that the assay used

to assess ACA in vitro does not predict complement

activation in vivo and, therefore, may not be a good

laboratory tool to evaluate the safety of antivenoms.

Alternatively, the test used to evaluate complement

activation in vivo, the CH50 assay, may not be of high

enough sensitivity to detect a minor activation of comp-

lement which may be pharmacologically significant for the

induction of EARs. For instance, high-dose intravenous

treatment of human immunoglobulin preparations induces

complement activation, demonstrated by increments in

serum C3bc, Bb and C5a, although no changes in

complement hemolytic activity, i.e. CH50, occurred

(Mollnes et al., 1998). Nevertheless, Montero et al. (1989)

demonstrated a reduction in CH50 in rabbits receiving a

whole IgG antivenom obtained by ammonium sulfate

precipitation, a procedure that yields a product with higher

content of aggregates and non-IgG proteins (Otero et al.,

1999). Additional studies aimed at detecting complement

activation in vivo, in both clinical and experimental settings,

are required to ascertain the value of in vitro ACA of

antivenoms in the prediction of EARs.

In conclusion, introduction of additional steps in the

production of whole IgG antivenoms, especially the

treatment with b-propiolactone after the purification of

horse IgG by caprylic acid precipitation, significantly

reduces in vitro ACA activity. If this test is indeed useful

in the assessment of the safety of these immunobiologicals

and in predicting EARs in patients, an antivenom produced

by caprylic acid precipitation and b-propiolactone treatment

would induce less EARs than an antivenom produced only

by caprylic acid precipitation. This hypothesis is currently

being tested in a clinical trial.

Acknowledgements

The authors thank Javier Nunez and the staff of

Production Division, Instituto Clodomiro Picado, for their

collaboration, and to G.H. Steinvorth for the donation of the

ion-exchange membrane. This study was supported by

Vicerrectorıa de Investigacion, Universidad de Costa Rica

(project 741-A1-027) and by UNESCO (grant 883.701-3).

This work was performed in partial fulfillment of

the doctoral degree of G. Leon at the University of Costa

Rica.

G. Leon et al. / Toxicon 45 (2005) 123–128 127

References

Angulo, Y., Estrada, R., Gutierrez, J.M., 1997. Clinical and

laboratory alterations in horses during immunization with

snake venoms for the production of polyvalent (Crotalinae)

antivenom. Toxicon 35, 81–90.

Barandun, S., Kistler, P., Jeunet, F., Isliker, H., 1962. Intravenous

administration of human g-globulin. Vox Sanguinis 7, 157–174.

Burnouf, T., Radosevich, M., 2000. Reducing the risk of infection

from plasma products: specific preventive strategies. Blood

Reviews 14, 94–110.

Cardoso, J.L.C., Fan, H.W., Franca, F.O.S., Jorge, M.T., Leite, R.P.,

Nishioka, S.A., Avila, A., Sano-Martins, I.S., Tomy, S.C.,

Santoro, M.L., Chudzinski, A.M., Castro, S.C.B.,

Kamiguti, A.S., Kelen, E.M.A., Hirata, M.H.,

Mirandola, R.M.S., Theakston, R.D.G., Warrell, D.A., 1993.

Randomized comparative trial of three antivenoms in the

treatment of envenoming by lance-headed vipers (Bothrops

jararaca) in Sao Paulo, Brazil. Quarterly Journal of Medicine

86, 315–325.

Chippaux, J.P., Lang, J., Amadi Eddine, S., Fagot, P., Rage, V.,

Peyrieux, J.C., Le Mener, V., 1998. Clinical safety of a

polyvalent F(ab0)2 equine antivenom in 223 African snake

envenomations: a field trial in Cameroon. Transactions of the

Royal Society of Tropical Medicine and Hygiene 92, 657–662.

Dart, R.C., McNally, J., 2001. Efficacy, safety and use of snake

antivenoms in the United States. Annals of Emergency

Medicine 37, 181–188.

Garcıa, M., Monge, M., Leon, G., Lizano, S., Segura, E., Solano, G.,

Rojas, G., Gutierrez, J.M., 2002. Effect of preservatives on IgG

aggregation, complement-activating effect and hypotensive

activity of horse polyvalent antivenom used in snakebite

envenomation. Biologicals 30, 143–151.

Gawarammana, I.B., Kularatne, S.A.M., Dissanayake, W.P.,

Kumarasiri, R.P.V., Senanayake, N., Ariyasena, H., 2004.

Parallel infusion of hydrocortisone G chlorpheniramine bolus

injection to prevent acute adverse reactions to antivenom for

snakebites. A randomized, double-blind, placebo-controlled

study. Medical Journal of Australia 180, 20–23.

Grandgeorge, M., Veron, J.L., Lutsch, C., Makula, M.F., Riffard, P.,

Pepin, S., Scherrman, J.M., 1996. Preparation of improved

F(ab 0)2 antivenoms. An example: new polyvalent European

viper antivenom (equine), in: Bon, C., Goyffon, M. (Eds.),

Envenomings and Their Treatments, Fondation Marcel Mer-

ieux, Lyon, pp. 161–172.

Guidolin, R., Morais, J.F., Stephano, M.A., Marcelino, J.R.,

Yamaguchi, I.K., Higashi, H.G., 1997. Effect of b-propiolactone

treatment on the complement activation mediated by equine

antisera. Revista Instituto de Medicina Tropical de Sao Paulo

39, 119–122.

Gutierrez, J.M., 1995. Clinical toxicology of snakebite in Central

America, in: Meier, J., White, J. (Eds.), Handbook of Clinical

Toxicology of Animal Venoms and Poisons. CRC Press,

Florida, pp. 645–665.

Jones, R.G.A., Landon, J., 2003. A protocol for ‘enhanced pepsin

digestion’: a step by step method for obtaining pure antibody

fragments in high yield from serum. Journal of Immunological

Methods 275, 239–250.

Jungi, T.W., Santer, M., Lerch, P.G., Barandun, S., 1986. Effect of

various treatments of gamma-globulin (IgG) for achieving

intravenous tolerance on the capacity to interact with human

monocyte Fc receptors. Vox Sanguinis 51, 18–26.

Lalloo, D.G., Theakston, R.D.G., 2003. Snake antivenoms. Journal

of Toxicology Clinical Toxicology 41, 277–290.

Leon, G., Monge, M., Rojas, E., Lomonte, B., Gutierrez, J.M., 2001.

Comparison between IgG and F(ab 0)2 polyvalent antivenoms:

neutralization of systemic effects induced by Bothrops asper

venom in mice, extravasation to muscle tissue, and potential for

induction of adverse reactions. Toxicon 39, 793–801.

Malasit, P., Warrell, D.A., Chanthavanich, P., Viravan, C.,

Mongkolsapaya, J., Singhthong, B., Supich, C., 1986. Predic-

tion, prevention, and mechanism of early (anaphylactic)

antivenom reactions in victims of snake bites. British Medical

Journal 292, 17–20.

Mollnes, T.E., Hogasen, K., de Carolis, C., Vaquero, E.,

Nielsen, E.W., Fontana, L., Perricone, R., 1998. High-dose

intravenous immunoglobulin treatment activates complement in

vivo. Scandinavian Journal of Immunology 48, 312–317.

Montero, J., Trejos, M., Lomonte, B., 1989. Efecto del suero

antiofıdico sobre la actividad hemolıtica del complemento

humano (in vitro) y de conejo (in vitro e in vivo). Revista

Costarricense de Ciencias Medicas 10, 1–9.

Morais, J.F., de Freitas, M.C.W., Yamaguchi, I.K., dos

Santos, M.C., Dias da Silva, W., 1994. Snake antivenoms

from hyperimmunized horses: comparison of the antivenom

activity and biological properties of their whole IgG and F(ab 0)2

fragments. Toxicon 32, 725–734.

Moran, N.F., Newman, W.J., Theakston, R.D.G., Warrell, D.A.,

Wilkinson, D., 1998. High incidence of early anaphylactoid

reaction to SAIMR polyvalent snake antivenom. Transactions of

the Royal Society of Tropical Medicine and Hygiene 92, 69–70.

Otero, R., Gutierrez, J.M., Rojas, G., Nunez, V., Dıaz, A.,

Miranda, E., Uribe, A.F., Silva, J.F., Ospina, J.G., Medina, Y.,

Toro, M.F., Garcıa, M.E., Leon, G., Garcıa, M., Lizano, S., De

La Torre, J., Marquez, J., Mena, Y., Gonzalez, N., Arenas, L.C.,

Puzon, A., Blanco, N., Sierra, A., Espinal, M.E., Arboleda, M.,

Jimenez, J.C., Ramırtez, P., Dıaz, M., Guzman, M.C., Barros, J.,

Henao, S., Ramırez, A., Macea, U., Lozano, R., 1999. A

randomized blinded clinical trial of two antivenoms, prepared

by caprylic acid or ammonium sulphate fractionation of IgG, in

Bothrops and Porthidium snake bites in Colombia: correlation

between safety and biochemical characteristics of antivenoms.

Toxicon 37, 895–908.

Otero-Patino, R., Cardoso, J.L.C., Higashi, H.G., Nunez, V.,

Sierra, A., Dıaz, A., Toro, M.F., Garcıa, M.E., Moreno, A.M.,

Medina, M.C., Castaneda, N., Silva-Dıaz, J.F., Murcia, M.,

Cardenas, S.Y., Dias da Silva, W.D., 1998. A randomized,

blinded, comparative trial of one-pepsin digested and two whole

IgG antivenoms for Bothrops snake bites in Uraba, Colombia.

American Journal of Tropical Medicine and Hygiene 58, 183–

189.

Rojas, G., Jimenez, J.M., Gutierrez, J.M., 1994. Caprylic acid

fractionation of hyperimmuine horse plasma: description of a

rapid procedure for antivenom production. Toxicon 32, 351–363.

Saetang, T., Treamwattana, N., Suttijitpaisal, P.,

Ratanabanangkoon, K., 1997. Quantitative comparison on the

refinement of horse antivenom by salt fractionation and ion-

exchange chromatography. Journal of Chromatography 700,

233–239.

Schosinsky, K., Vargas, M., Vinocour, G., Gonzalez, O.M.,

Brilla, E., Gutierrez, A., 1983. Manual de Tecnicas de

G. Leon et al. / Toxicon 45 (2005) 123–128128

Laboratorio. Quımica Clınica. Universidad de Costa Rica, San

Jose p. 224.

Stephan, W., 1975. Undegraded human immunoglobulin for

intravenous use. Vox Sanguinis 28, 422–437.

Sutherland, S.K., 1977. Serum reactions. An analysis of commercial

antivenoms and the possible role of anticomplementary activity

in de-novo reactions to antivenoms and antitoxins. Medical

Journal of Australia 1, 613–615.

Tanaka, K., Sawatani, E., Dias, G.A., Shigeoka, E.M.,

Campos, T.C.X.B., Nakao, H.C., Arashiro, F., 2000. High

quality human immunoglobuin G purified from Cohn fractions

by liquid chromatography. Brazilian Journal of Medical and

Biological Research 33, 27–30.

Theakston, R.D.G., Warrell, D.A., Griffiths, E., 2003. Report of a

WHO workshop on the standardization and control of

antivenoms. Toxicon 41, 541–557.