the assay must be done as soon as the sample thaws. Ideal
processing and storage conditions did occur with the rat
samples because of the controlled experimental condi-
tions. The results demonstrated similar measured venom
concentrations with minimal spread of the data, similar to
those fou nd by previous investigators. (Barral-Netto et al.,
1990) Repeating the assay after further freeze-thawing
cycles may result in unreliable results and should only be
used to conﬁrm the presence of venom in the sample, but
not quantify the amount.
This study describes a highly sensitive and reliable assay
for the detection and quantiﬁcation of venom in blood
incorporating the biotin-streptavidin ampliﬁcation and the
antivenom difference technique. This method is appropriate
for all Australian snake venoms because of the expected low
concentrations that may be present, particularly if there is
a delay from the bite to the time of the blood sample.
Although the LoD for the assay was 0.15 ng/mL all sample
results less than 1 ng/mL should be repeated with the
antivenom difference method (LoD 0.1 ng/mL) and for other
snake venoms, if appropriate.
We wish to acknowledge the many clinicians and labo-
ratory staff involved in recruitment and collection of blood
samples for analysis as part of the Australian Snakebite
Project and in particular for cases recruited here by Chris
Barnes (Bundaberg Hospital), Robert Bonnin, Richard Whi-
taker and Lambros Halkidis (Cairns Base Hospital), Kate
Porges (Gosford Hospital), Todd Fraser (Mackay Base
Hospital), Bart Currie (Royal Darwin Hospital), Tanya Gray
(Royal Children’s Hospital Brisbane) and Julian White
(Women’s and Children’s Hospital, Adelaide) and clinical
toxicologists around Australia at the poison information
centres for referring additional cases.
Conﬂict of interest
The study was supported in part by NHMRC Project
Grant 490305. GKI is supported by an NHMRC Clinical
Career Development Award ID605817 and SGAB is sup-
ported by NHMRC Clinical Career Development Award
Ariaratnam, C.A., Meyer, W.P., Perera, G., et al., 1999. A new monospeciﬁc
ovine Fab fragment antivenom for treatment of envenoming by the
Sri Lankan Russell’s viper (Daboia Russelii Russelii): a preliminary
dose-ﬁnding and pharmacokinetic study. American Journal of Trop-
ical Medicine and Hygiene 61, 259–265.
Audebert, F., Grosselet, O., Sabouraud, A., Bon, C., 1993. Quantitation of
venom antigens from European vipers in human serum or urine by
ELISA. Journal of Analytical Toxicology 17, 236–240.
Barral-Netto, M., Schriefer, A., Barral, A., Almeida, A.R., Mangabeira, A.,
1991. Serum levels of bothropic venom in patients without antivenom
intervention. American Journal of Tropical Medicine and Hygiene 45,
Barral-Netto, M., Schriefer, A., Vinhas, V., Almeida, A.R., 1990. Enzyme-
linked immunosorbent assay for the detection of Bothrops jararaca
venom. Toxicon 28, 1053–1061.
Brunda, G., Sashidhar, R.B., Sarin, R.K., 2006. Use of egg yolk antibody
(IgY) as an immunoanalytical tool in the detection of Indian cobra
(Naja naja naja) venom in biological samples of forensic origin. Tox-
icon 48, 183–194.
Coulter, A.R., Cox, J.C., Sutherland, S.K., Waddell, C.J., 1978. A new solid-
phase sandwich radioimmunoassay and its application to the detec-
tion of snake venom. Journal of Immunological Methods 23, 241–252.
Coulter, A.R., Sutherland, S.K., Broad, A.J., 1974. Assay of snake venoms in
tissue ﬂuids. Journal of Immunological Methods 4, 297–300.
Dong, L.V., Quyen, L.K., Eng, K.H., Gopalakrishnakone, P., 2003. Immuno-
genicity of venoms from four common snakes in the South of Viet-
nam and development of ELISA kit for venom detection. Journal of
Immunological Methods 281, 13–31.
Gan, M., O’Leary, M.A., Brown, S.G., et al., 2009. Envenoming by the rough-
scaled snake (Tropidechis carinatus): a series of conﬁrmed cases.
Medical Journal of Australia 191, 183–186.
Guo, M.P., Wang, Q.C., Liu, G.F., 1993. Pharmacokinetics of cytotoxin from
Chinese cobra (Naja naja atra) venom. Toxicon 31, 339–343.
Ho, M., Warrell, D.A., Looareesuwan, S., et al., 1986. Clinical signiﬁcance of
venom antigen levels in patients envenomed by the Malayan pit viper
(Calloselasma rhodostoma). American Journal of Tropical Medicine
and Hygiene 35, 579–587.
Isbister, G.K., Brown, S.G., MacDonald, E., White, J., Currie, B.J., 2008.
Current use of Australian snake antivenoms and frequency of
immediate-type hypersensitivity reactions and anaphylaxis. Medical
Journal of Australia 188, 473–476.
Isbister, G.K., Halkidis, L., O’Leary, M.A., et al., 2010a. Human anti-snake
venom IgG antibodies in a previously bitten snake-handler, but no
protection against local envenoming. Toxicon 55, 646–649.
Isbister, G.K., Little, M., Cull, G., et al., 2007a. Thrombotic microangiopathy
from Australian brown snake (Pseudonaja) envenoming. Internal
Medicine Journal 37, 523–528.
Isbister, G.K., O’Leary, M.A., Hagan, J., et al., 2010b. Cross-neutralisation of
Australian brown snake, taipan and death adder venoms by mono-
valent antibodies. Vaccine 28, 798–802.
Isbister, G.K., O’Leary, M.A., Schneider, J.J., Brown, S.G., Currie, B.J., 2007b.
Efﬁcacy of antivenom against the procoagulant effect of Australian
brown snake (Pseudonaja sp.) venom: in vivo and in vitro studies.
Toxicon 49, 57–67.
Meyer, W.P., Habib, A.G., Onayade, A.A., et al., 1997. First clinical experi-
ences with a new ovine Fab Echis ocellatus snake bite antivenom in
Nigeria: randomized comparative trial with Institute Pasteur Serum
(Ipser) Africa antivenom. American Journal of Tropical Medicine &
Hygiene 56, 291–300.
NCCLS, 2004. Protocols for Determination of Limit of Detection and Limit
of Quantitation; Approved Guideline. NCCLS document EP17-A.
Norris, R.L., Pfalzgraf, R.R., Laing, G., 2009. Death following coral snake
bite in the United States – ﬁrst documented case (with ELISA
conﬁrmation of envenomation) in over 40 years. Toxicon.
O’Leary, M.A., Brown, S.G.A., Jacoby, T., Gan, M., Schneider, J., Isbister, G.K.,
2008. A sensitive and speciﬁc enzyme immunoassay to distinguish
between two closely related Australian elapids: Tropidechis carinatus
and Notechis scutatus. In: 8th Asia-Paciﬁc Congress on Animal, Plant
and Microbial Toxins. International Society of Toxinology, Vietnam.
O’Leary, M.A., Isbister, G.K., Schneider, J.J., Brown, S.G., Currie, B.J., 2006.
Enzyme immunoassays in brown snake (Pseudonaja spp.) envenom-
ing: detecting venom, antivenom and venom-antivenom complexes.
Toxicon 48, 4–11.
Otero, R., Leon, G., Gutierrez, J.M., et al., 2006. Efﬁcacy and safety of two
whole IgG polyvalent antivenoms, reﬁned by caprylic acid fraction-
ation with or without beta-propiolactone, in the treatment of
Bothrops asper bites in Colombia. Transactions of the Royal Society of
Tropical Medicine and Hygiene 100, 1173–1182.
Pardal, P.P.D., Souza, S.M., Monteiro, M.R.D.D., et al., 2004. Clinical trial of
two antivenoms for the treatment of Bothrops and Lachesis
eastern Amazon region of Brazil. Transactions of the Royal
Society of Tropical Medicine and Hygiene 98, 28–42.
Rebeski, D.E., Winger, E.M., Shin, Y.K., et al., 1999. Identiﬁcation of unac-
ceptable background caused by non-speciﬁc protein adsorption to the
plastic surface of 96-well immunoassay plates using a standardized
enzyme-linked immunosorbent assay procedure. Journal of Immu-
nological Methods 226, 85–92.
Selvanayagam, Z.E., Gopalakrishnakone, P., 1999. Tests for detection of
snake venoms, toxins and venom antibodies: review on recent trends
(1987–1997). Toxicon 37, 565–586.
S. Kulawickrama et al. / Toxicon 55 (2010) 1510–1518 1517