Article

Development of a sensitive enzyme immunoassay for measuring taipan venom in serum

Emergency Department, Gold Coast Hospital, Queensland, Australia.
Toxicon (Impact Factor: 2.49). 03/2010; 55(8):1510-8. DOI: 10.1016/j.toxicon.2010.03.003
Source: PubMed

ABSTRACT

The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.

Full-text

Available from: Wayne C Hodgson
Development of a sensitive enzyme immunoassay for measuring taipan
venom in serum
S. Kulawickrama
a
, M.A. O’Leary
b
, W.C. Hodgson
c
, S.G.A. Brown
d
,
e
,
T. Jacoby
d
,
f
, K. Davern
f
, G.K. Isbister
b
,
g
,
h
,
*
a
Emergency Department, Gold Coast Hospital, Queensland, Australia
b
Department of Clinical Toxicology and Pharmacology, Calvary Mater Hospital, Newcastle, NSW, Australia
c
Monash Venom Group, Department of Pharmacology, Monash University, VIC, Australia
d
Centre for Clinical Research in Emergency Medicine, Western Australian Institute for Medical Research and
the University of Western Australia Centre for Medical Research, WA, Australia
e
Emergency Medicine, Royal Perth Hospital, University of Western Australia, WA, Australia
f
Monoclonal Antibody Facility, Western Australian Institute for Medical Research and
the University of Western Australia Centre for Medical Research, WA, Australia
g
Discipline of Clinical Pharmacology, University of Newcastle, NSW, Australia
h
Tropical Toxinology Unit, Menzies School of Health Research, Darwin, NT, Australia
article info
Article history:
Received 26 January 2010
Received in revised form
27 February 2010
Accepted 3 March 2010
Available online 16 March 2010
Keywords:
Taipan venom
Snake envenoming
Enzyme-imunnoassays
Antivenom
abstract
The detection and measurement of snake venom in blood is impo rtant for confirming
snake id entification, determining w hen sufficient antivenom has been given, detecting
recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays
(EIA) have had persistent problems with poor sensitivity and high background absor-
bance leading to fa lse positive results. This is particularly problematic with Australasian
snakes where small amounts of highly potent venom are injected, resulting in low
concentrations being associated with severe clinical effects. We aimed to develop
a venom EIA with a limit of detection ( LoD) sufficient to accurately distinguish mild
envenoming from b ackground absorbance at picogram concentrations of venom in blood.
Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before
and after antivenom, and from rats given known venom doses. A sandwich EIA was
developed using biotinylated rabbit anti-sn ake venom antibodies for detection. For low
venom concent rations (i.e. <1 ng/mL) the assay was done before and after addit ion of
antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the
standard assay and 0.1 ng /mL for the antivenom difference method. In 11 pre-antivenom
samples the median venom concentration was 10 ng/mL (Range: 0.3–3212 ng /mL). In four
patients with in complete venom-induced con sumption coagulopathy th e median venom
concentration was 2.4 ng/mL compared to 30 ng/mL in seven patient s with compl ete
venom-induced consumption coagulopathy. No venom was detected in any post-
antivenom sample and the median antivenom dose prior to this first post-antivenom
sample was 1.5 vials (1–3 vials), includi ng 7 patients administered only 1 vial. In rats
the assay distinguished a 3-fold difference in venom dose administered and there was
small inter-individual variability. There was small but measurable cross-reactivity with
black snake (Pseudechis), tiger snake (Notechis ) and rough-scale snake (Tropidechis car-
inatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of
*
Corresponding author. University of Newcastle, c/o Calvary Mater Newcastle, Edith St, Waratah NSW 2298, Australia. Tel.: þ612 4921 1211; fax: þ612
4921 1870.
E-mail address: geoff.isbister@gmail.com (G.K. Isbister).
Contents lists available at ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
0041-0101/$ see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2010.03.003
Toxicon 55 (2010) 1510–1518
Page 1
biotinylation and the antivenom dif ference method in venom EIA produces a highly
sensitive assay that will be us eful for determining antiven om dose, forensic and clinical
diagnosis.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
The detection and measurement of snake venom in blood
is important in clinical toxinology and toxinology research
for diagnosis and assessment of antivenom effectiveness,
but is not often available. Measurement of (snake) venom in
human and animal tissue and serum was first undertaken
with solid-phase radioimmunoassay. (Coulter et al., 1974)
Subsequently, enzyme immunoassays were developed by
Theakston et al. (1977) with some refinement over the last
30 years. (Selvanayagam and Gopalakrishnakone, 1999)
Venom enzyme immunoassays have been used to confirm
envenoming by particular species in studies of comparative
effectiveness of different antivenom preparations, (Meyer
et al., 1997; Pardal et al., 2004; Otero et al., 2006) studies
of antivenom dosing, (Theakston et al., 1992; Ariaratnam
et al., 1999) forensic and diagnostic studies, (Brunda et al.,
2006; Isbister et al., 2010a; Gan et al., 2009; Norris et al.,
2009) and in assessment of first aid effectiveness in
retarding venom absorption (Tun et al., 1995).
A particular problem with venom enzyme immunoas-
says has been high background absorbance which, in some
cases, has lead to the incorrect interpretation of non-
envenomed cases. Background interference is a well rec-
ognised problem with enzyme immunoassays, (Rebeski
et al., 1999) and previous reports of persistent venom
antigenaemia (Ariaratnam et al., 1999; Ho et al., 1986)
are more likely due to high background in these samples,
rather than the postulated recurrence or inefficacy of
antivenom in binding venom. This is a significant issue
because a central role for venom enzyme immunoassays is
to determine when sufficient antivenom has been admin-
istered and this outcome is dependent on the assay being
able to distinguish low concentrations of venom from no
venom. A number of approaches have been developed to
reduce background absorbance, including using normal
population blanks (Ho et al.,1986), measurement before and
after the addition of antivenom (O’Leary et al., 2006) and
using biotin-avidin amplification. (Dong et al., 2003; Guo
et al., 1993; Selvanayagam and Gopalakrishnakone, 1999)
Limits of detection (LoD) for venom or toxin assays have
been reported between 0.2 and 10 ng/mL, (Audebert et al.,
1993; Sjostrom et al., 1996; Guo et al., 1993; Barral-Netto
et al., 1991), depending on the use of these methods to
reduce background absorbance.
Developing an assay with a good LoD is especially
important for Australian elapids because they have short
fangs and highly potent venom, which can result in severe
envenoming being associated with low serum venom
concentrations. This was found in a previous assay devel-
oped which only had a LoD of 4 ng/mL and was unable to
detect venom in some brown snake (Pseudechis spp.)
envenomings with significant coagulopathy. (O’Leary et al.,
20 06).
Taipan (Oxyuranus spp.) envenoming is important in
northern Australia and Papua New Guinea and no previous
studies have measured taipan venom in patient samples.
There is continuing controversy over the dose of antivenom
required for taipan envenoming (White, 2001) and what
end points should be used to determine this. Developing an
assay to measure taipan venom concentrations in enve-
nomed patients is required so that the antivenom dose can
be determined, similar to previous studies on Australian
brown snake (Pseudechis spp.) (Isbister et al., 2007b) and
rough-scale snake (Tropidechis carinatus). (
Gan et al., 2009).
The
aim
of this study was to develop a highly sensitive
venom enzyme immunoassay to measure taipan venom in
human and animal serum that is accurate enough to
distinguish envenoming from background absorbance at
picogram venom concentrations. In addition, the study
aimed to provide an approach to defining LoD in venom
enzyme immunoassays, evaluating the effect of sample
processing and storage and the cross-reactivity of the assay
with similar snake venoms.
2. Materials and methods
2.1. Materials
Coastal taipan (Oxyuranus scutellatus), common brown
snake (Pseudonaja textilis), tiger snake (Notechis scutatus),
rough-scale snake (T. carinatus), Stephen’s banded snake
(Hoplocephalus stephensii), mulga snake (Pseudechis aus-
tralis), red-bellied black snake (Pseudechis porphyriacus),
death adder (Acanthophis antarcticus) and Malaysian pit
viper (Calloselasma rhodostoma) venoms were purchased
from Venom Supplies, Tanunda, South Australia. All venoms
used are pooled from more than one snake milked on
multiple occasions. Tetramethylbenzidine (TMB), rabbit
anti-Horse IgG peroxidase conjugate, standard serum
(#7023) and bovine serum albumin (BSA) were obtained
from Sigma. All chemicals used were of analytical grade. PBS
is phosphate-buffered saline. Plates were read at 450 nm on
a BioTek ELx808 plate reader. Rabbit IgG antibodies were
biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce #
21335). Streptavidin-conjugated horseradish peroxidase
was obtained from Millipore. Taipan antivenom (TAV)
(Batch # 0548-05601) was obtained from CSL Ltd. All
procedures were carried out at room temperature.
2.1.1. Rabbit anti-snake venom monovalent antibodies
Polyclonal monovalent rabbit IgG to coastal taipan
(O. scutellatus) venom was prepared as previously described.
(Isbister et al., 2010b) In brief, rabbits were injected with
coastal taipan venom emulsified with Freund’s adjuvant,
boosted 4 weeks later with the same venom concentration
in incomplete Freund’s adjuvant, and then 10–20 mL of
blood was collected from the rabbit. Rabbits had ongoing
S. Kulawickrama et al. / Toxicon 55 (2010) 1510–1518 151 1
Page 2
boosters for further blood collection. Indirect enzyme
immunoassay was used to confirm the presence of anti-
snake venom antibodies, and if there were antibody titres
higher than 50,000, IgG was purified with affinity purifi-
cation by being passed through a Sepharose-Protein G
column using a BioRad BioLogic liquid chromatography
system.
2.2. Samples
Envenomed and non-envenomed humans and enve-
nomed rat serum samples were obtained for the assay. The
use of rat samples provided a controlled situation where
a known amount of venomwas administered, the processing
time was short and samples were all analysed after freezing
once. Spiked samples were used to investigate the effect of
different storage conditions on the venom concentration
determination compared to immediate measurement and to
do cross-reactivity experiments.
2.2.1. Human samples
Serum samples were obtained as part of the Australian
Snakebite Project (ASP), a multicentre study of snakebite
from over 100 hospitals (Isbister et al., 2008). Participants
have demographic information, clinical features, laboratory
results and treatments recorded on a clinical research form
which is then entered in a relational database. Ethics
approval has been obtained from eighteen Human
Research and Ethics Committees covering all institutions
involved in the study. Serum or plasma samples were
available from 17 patients with suspected or confirmed
taipan bites (Tables 1 and 2). Samples before and after
antivenom were included for analysis if available. Two
hundred and twenty nine samples from patients with
suspected snake envenoming and no clinical or laboratory
features of envenoming were used as blanks and for low
concentration venom spiking studies. All samples were
stored for 6 months–4 years at 80
C until assayed.
2.2.2. Rat samples
Male Sprague–Dawley rats were anaesthetised using
pentobarbitone sodium (60–100 mg/kg, i.p.). Additional
Table 1
Demographic information, clinical effects, snake identification, antivenom treatment, venom and antivenom concentrations for each of the human cases of
taipan envenoming.
Sex/age Venom concentration
(ng/mL)
AV concentration
(mU/mL)
First AV dose
a
Total AV
dose
Clinical effects Snake ID
M34 2.8 16684 1 PVAV 1 PVAV VICC (mild) Coastal Taipan
3 TAV
M66 3212 17424 3 PVAV 3 PVAV VICC þ NT Coastal Taipan
1 TAV
M38 6.1 1720 1 TAV 2 TAV VICC Coastal Taipan
M45 10 6584 1 PVAV 1 PVAV VICC þ NT (mild) Inland Taipan
1 TAV
M36 30 9224 2 TAV 4 TAV VICC þ NT Coastal Taipan
(Hospital ID only)
M23 1.9 6022 1 PVAV þ 2 TAV 1 PVAV VICC (mild) þ VA Coastal Taipan
2 TAV
M50 NA 2832 1 TAV 1 TAV VICC(mild) þ VA Coastal Taipan
M19 NA 35084 1 TAV 5 TAV VICC þ NT Coastal Taipan
4 TSAV
F55 <LoD NA NA Nil Nil Coastal Taipan
M48 232 21309 2 TAV 2 TAV VICC þ NT þ TM Coastal Taipan
M47 165 7864 2 PVAV 2 PVAV VICC þ NT Coastal Taipan (No ID)
2 TAV
M44 1.9 4599 1 PVAV 2 PVAV VICC (mild) Coastal Taipan (No ID)
F12 16 14017 2 (PVAV/TAV) 1 PVAV VICC þ NT(mild) Coastal Taipan (No ID)
1 TAV
F69 NA 4268 1 PVAV 1 PVAV VICC(mild) þ TM Coastal Taipan (No ID)
1 TAV
M22 NA 6670 2 (PVAV/TAV) 1 PVAV VA Coastal Taipan
1 TAV
M31 <LoD NA Nil Nil Nil Coastal Taipan
M39 8.4 18727 3 (PVAV/2TAV) Nil NT(mild) þ VICC(mild) Coastal Taipan (No ID)
AV ¼ antivenom; PVAV ¼ polyvalent antivenom; TAV ¼ taipan antivenom; VICC ¼ venom-induced consumption coagulopathy; NT ¼ neurotoxicity;
TM ¼ thrombotic microangiopathy characterised by thrombocytopaenia, acute renal failure and microangiopathic haemolytic anaemia; (Isbister et al.,
2007a)VA¼ venom allergy; ID ¼ identification; LoD ¼ limit of detection; NA ¼ not available.
a
Dose given prior to the first blood sample taken post-antivenom.
Table 2
Venom concentration measurements (ng/mL) in the same sample, on
multiple occasions.
Patient Initial results
a
Second assay Third assay Fourth assay
M23 1.9 0.7
M48
b
152 146 59 50
M45 10 5.5 4.1
M47 165 144 52
M66 3212 565
F12 16 6.4
M36 30 9
a
The initial assay was always done from specimens that were collected
from the patient, separated and frozen once. However, the time from
collection to the first freezing was variable.
b
These results are from the second sample so differ from Table 1.
S. Kulawickrama et al. / Toxicon 55 (2010) 1510–15181512
Page 3
anaesthetic was administered throughout the experiment
as required. An incision was made in the cervical region,
and cannulae were placed in the right jugular vein (for
venom administration), the carotid artery (for blood
sampling) and the trachea (for artificial respiration, if
required). After allowing the preparation to stabilise for at
least 20 min after surgery, a blood sample (0.5 mL) was
collected in a MiniCollect 0.5 mL LH/Gel separation tube
(Greiner bio-one). Two venom doses were used five rats
were injected with 2
m
g/kg of taipan venom and another
5 with 6
m
g/kg. Blood samples were taken 5, 15, 30, 60 and
120 min post-venom administration. Immediately after
collection, the samples were spun at 6500 rpm for 15 min,
the serum removed and frozen. All experiments were
approved by the Monash University (SOBS-B) animal ethics
committee.
2.2.3. Spiked samples under different storage conditions
Undiluted samples from twenty-four non-envenomed
patients were spiked with taipan venom at 50 ng/mL and
then divided into four aliquots. The first aliquot was
assayed immediately. The second aliquot was stored in the
refrigerator (4
C) for 3 days. The third and fourth aliquots
were immediately frozen for 3 days, the fourth being
thawed and refrozen once during that time. Venom assays
were done on aliquots 2–4 on the same day under the same
conditions.
2.3. Enzyme immunoassay
2.3.1. Venom enzyme immunoassay
Greiner Microlon 96-well high-binding plates were
coated with rabbit anti-taipan IgG (1
m
g/mL) in carbonate
buffer (50 mM, pH 9.6), kept at room temperature for 1 h
and then at 4
C overnight. The plates were then washed
once with PBS containing 0.02% TWEEN 20, and 30 0
m
Lof
blocking solution of 0.5% BSA in PBS was added. After 1 h
the plates were washed again, and 100
m
L of sample solu-
tion (human, rat, spiked [taipan venom] or blank sample)
was applied as a 0.1%–10% dilution in PBS or blocking
solution. The plates were allowed to stand for 1 h and then
washed three times. Next, biotinylated anti-taipan IgG
(100
m
L, 0.15
m
g/mL in blocking solution) was added. After
standing for a further hour the plates were washed again.
Streptavidin HRP (100
m
L, 1
m
g/mL in blocking solution) was
added and left for 1 h. The plate was then washed four
times and 100
m
L of TMB reagent added and colour allowed
to develop for 4–10 min. The reaction was stopped by the
addition of 50
m
Lof1MH
2
SO
4
. All samples were measured
in triplicate, and the averaged absorbances converted to
a concentration by comparison with a standard curve based
on serial dilutions of venom, using a linear equation (for
values <2 ng/mL) or, for higher values, a sigmoidal curve.
2.3.2. Determination of the limit of blank (LoB)
The limit of blank (LoB) was determined according to
the EP17-A protocol. (NCCLS, 2004) The EP17-A, in line with
other international consensus statements, defines the LoB
as the 95th percentile value of the distribution of blank
values; i.e. the limit for declaring a measured value signif-
icantly higher than the blank. (NCCL S, 2004) A total of 229
samples were assayed from 57 non-envenomed patient
samples on ten different days. The distribution of the
measured values was examined to determine if it was
parametric and the LoB was then determined according to
whether the blank values had a parametric distribution
or not.
2.3.3. Determination of the limit of detection (LoD)
The limit of detection (LoD) was also determined
according to the EP17-A protocol (NCCLS, 2004) and
defined as the actual sample concentration (spiked with
taipan venom) where 95% of the measured concentrations
were above the LoB. A similar number of low concentration
spiked samples were assayed as for the LoB initially using
a spiking concentration of four times the LoB. Depending
on the distribution of the LoD and the spread of the data,
either parametric or non-parametric methods were used. If
the data were non-parametric and the variance differed at
different venom concentrations a trial and error approach
was used. In this case blank samples were spiked with
lower and lower concentrations until more than 5% of the
spiked samples had measured concentrations below the
LoB. The LoD was then the lowest concentration when less
than 5% of the measured concentrations were below the
LoB. Full details are available in the EP17-A document.
(NCCLS, 2004).
2.3.4. Antivenom difference method for low venom
concentrations (<1 ng/mL)
To improve the LoD for low venom concentrations,
samples were assayed before and after the addition of
commercial taipan antivenom. This method has previously
been described to decrease non-specific background
absorbance in a brown snake (Pseudechis) venom assay.
(O’Leary et al., 2006)100
m
L of TAV was added to 800
m
L
PBS. This solution was divided and TAV (50
m
L, 25 mU) was
added to one half and PBS (50
m
L) to the other. These
solutions were allowed to stand for 1 h then applied to the
plate. The difference in the measured venom concentra-
tions with and without antivenom is taken as the final
measured venom concentration. This procedure was done
for both blank and spiked samples. LoB and LoD were
determined for this difference method using the same
procedure as described above.
2.3.5. Cross-reactivity with other snake venoms
Serial dilutions of the eight non-taipan snake venoms in
blocking solution were prepared to produce a series of
concentrations for each venom of 2.5, 1.25, 0.62, 0.31, 0.15
and 0.08 ng/mL. These were applied to the plate instead of
samples with taipan venom. The taipan venom enzyme
immunoassay was then used on these samples as described
above.
2.3.6. Antivenom enzyme immunoassay
Antivenom was measured in serum samples using
a previously developed method. (O’Leary et al., 2006)In
brief, plates were coated with taipan venom (1
m
g/mL in
carbonate buffer) for 1 h at room temperature then at 4
C
overnight. After washing and blocking, samples were
applied at a dilution of 0.05–0.1% in PBS and standards at
S. Kulawickrama et al. / Toxicon 55 (2010) 1510–1518 1513
Page 4
0–20 mU/mL in PBS. Detection was achieved with rabbit
anti-Horse IgG peroxidase conjugate (0.5
m
g/mL in blocking
solution), followed by treatment with TMB/H
2
SO
4
.
2.4. Data analysis
Standard curves were fitted by linear and non-linear
regression. Normality of the data was assessed by the
Kolmogorov–Smirnov test and the Shapiro–Wilk normality
test. All analyses and graphics were done in GraphPad
Prism version 5.01 for Windows, GraphPad Software, San
Diego California USA, www.graphpad.com.
3. Results
3.1. Determination of venom assay sensitivity LoB and LoD
The measured venom concentrations in 229 assays of
blank (i.e. non-envenomed) samples are plotted as a histo-
gram in Fig. 1A–D. The measured concentrations were not
normally distributed so the 95th percentile was taken as
the LoB and was equal to 0.07 ng/mL. Spiked samples
ranging from 0.5 to 2 ng/mL were measured on multiple
occasions and demonstrated that the distributions of the
measured venom concentrations were not normal and the
variance increased with increasing concentration (data not
shown). A non-parametric trial and error approach was
then undertaken to determine the LoD.
Twenty-four to thirty two blank samples were spiked
with taipan venom at concentrations of 0.5, 0.3, 0.15 and
0.1 ng/mL to determine the LoD. For spiked venom
concentrations of 0.5, 0.3 and 0.15 ng/mL there was sepa-
ration of the distributions of the blank samples and the
spiked samples (Fig. 1A,B,C), but at a spiked concentration
of 0.1 ng/mL more than 5% of the measured concentrations
were below the LoB (Fig. 1D). Therefore the LoD was
determined to be 0.15 ng/mL.
3.2. Antivenom difference method for venom assay
Thirty two samples at the lower spiked taipan venom
concentrations of 0.15 and 0.1 ng/mL were assayed with
and without the addition of commercial taipan antivenom
and the difference used as the measured venom concen-
tration. The addition of antivenom did not decrease the
background in non-spiked non-envenomed samples and
the LoB for this method was 0.04 ng/mL. The LoD was
0.1 ng/mL using the same procedure as above (Fig. 2).
3.3. Human samples
The results of the venom assays for human samples are
detailed in Table 1. Pre-antivenom samples were only
available in 13 of the 17 patients. Two patients had no
evidence of envenoming and venom was not detectable in
any sample from these patients. In the 11 pre-antivenom
samples the median venom concentration was 10 ng/mL
(Range: 0.3–3212 ng/mL). In four patients with incomplete
venom-induced consumption coagulopathy the median
venom concentration was 2.4 ng/mL compared to a median
venom concentration of 30 ng/mL in the seven patients with
complete venom-induced consumption coagulopathy. The
Fig. 1. Histograms of the measured venom concentrations for 229 blank samples (grey bars) compared to the measured venom concentration in samples spiked
with venom (black bars) at concentrations of 0.5 ng/mL (A, 141 samples), 0.3 ng/mL (B, 24 samples), 0.15 ng/mL (C, 32 samples) and 0.1 ng/mL (D, 30 samples).
S. Kulawickrama et al. / Toxicon 55 (2010) 1510–15181514
Page 5
median time until antivenom administration was similar for
incomplete venom-induced consumption coagulopathy (4 h
and 10 min) and complete venom-induced consumption
coagulopathy (3 h and 10 min). Post-antivenom samples
were available in all 15 patients administered antivenom
and no taipan venom could be detected in any sample. The
median dose of antivenom administered prior to the first
post-antivenom sample was 1.5 vials (1–3 vials), including
seven patients administered only 1 vial.
3.4. Rat samples
Plots of venom concentration versus time for rats
injected with intravenous taipan venom show that the
assay can distinguish between the two groups of rats
administered a 3-fold difference in venom dose and there is
only a small inter-individual variability in the assay values
in rats administered the same doses (Fig. 3).
3.5. Effect of storage conditions on venom assay
Stored samples were measured at 20–30% less than
freshly-spiked samples, and the method of storage made
little difference to the measurement. Samples from seven
patients were assayed on further occasions, with a freeze–
thaw cycle between each assay, and there was a significant
decrease in measured venom concentration on subsequent
assays (Table 3). In three samples the assay was repeated
without freeze thawing within 48 h and there was less
change in the measured venom concentration (data not
shown).
3.6. Cross-reactivity with other snake venoms
Table 3 summarises the results of the taipan assay for
the other eight snake venoms. There was negligible
detection of common brown snake (P. textilis), death adder
(A. antarcticus), Stephen’s banded snake (H. stephensii) and
Malaysian pit viper (C. rhodostoma) venoms by the taipan
venom enzyme immunoassay. There was measurable
cross-reactivity of the two black snake venoms (P. australis
and P. porphyriacus), tiger snake (N. scutatus) and rough-
scale snake (T. carinatus) venoms on the taipan assay. The
largest amount of cross-reactivity occurred between mulga
snake (P. australis) venom where a sample containing
0.8 ng/mL of mulga venom would register as 0.1 ng/mL on
the taipan assay. However, for measurements of 1 ng/mL on
the taipan assay, much larger amounts of the other snake
Fig. 2. Histograms of the measured venom concentrations using the difference of the assay with and without antivenom in 62 blank samples (grey bars)
compared to the measured venom concentration in samples spiked with venom (black bars) at concentrations of 0.15 ng/mL (A, 32 samples) and 0.1 ng/mL (B,30
samples).
Fig. 3. Measured venom concentrations versus time for rats administered
intravenous venom 2 mg/kg (N ¼ 5, thick black line) and 6 mg/kg (N ¼ 5,
dashed line). Error bars represent the interquartile range around the median.
Table 3
Cross reactivity of the taipan enzyme immunoassay with other snake
venoms. Samples were spiked with various other snake venoms and then
detected using anti-taipan antibodies.
Snake venom Minimum
concentration
of venom measured
as >0.1 ng/mL (LoD)
taipan venom
Minimum
concentration
of venom measured
as >1 ng/mL taipan
venom
Mulga snake 0.8 25
Tiger snake 1.6 25–50
Red-bellied
black snake
6.2 >50
Rough-scale snake 6.2 >50
Stephen’s
banded snake
12.5 >>
Brown snake >12.5 >>
Death adder >12.5 >>
Malaysian pit viper >12.5 >>
S. Kulawickrama et al. / Toxicon 55 (2010) 1510–1518 1515
Page 6
venoms were required. In the case of mulga snake venom,
25 ng/mL was required to register as 1 ng/mL on the assay.
3.7. Antivenom concentration
Antivenom was detected in all samples post-antivenom
dose and the values are provided in Table 1.
4. Discussion
This study describes a highly sensitive venom enzyme
immunoassay for taipan venom that has a LoD of 0.1 ng/mL.
The method can be applied to other venom enzyme
immunoassay and the addition of the biotinylation step to
the original assay is straightforward and inexpensive. This
approach will avoid previous problems with background
absorbance and allow accurate studies to define recurrence
of envenoming. The study also shows that storage condi-
tions affect the assay and that separation and freezing of
serum should be undertaken rapidly. In the rat experiments
under controlled conditions, the assay measured similar
concentrations of venom in blood for the same injected dose
in different rats. This suggests that under ideal dosing and
collection conditions, the assay has good reproducibility.
Previous venom enzyme immunoassays developed for
Australian snakes have not been sensitive enough to detect
low venom concentrations that can still be associated with
significant envenoming. The commercial snake venom
detection kit (CSL Ltd.) has not been validated for use with
blood samples and the kit is designed to distinguish
between the five major groups of snakes rather than detect
low venom concentrations. (Coulter et al., 1978) A previous
assay developed for brown snake venom (Pseudechis spp)
only had a LoD of 4 ng/mL, despite the use of the antivenom
difference method. (O’Leary et al., 2006) Table 1 demon-
strates that, at least for taipan bites, venom concentrations
below 4 ng/mL are associated with clinically significant
envenoming, including partial venom-induced consump-
tion coagulopathy.
Part of the success of this assay was the use of biotin-
streptavidin amplification to increase sensitivity, rather
than the standard peroxidase enzyme immunoassay system,
where the horseradish peroxidase is conjugated directly to
the detecting antibody. The use of biotin-avidin (or biotin-
streptavidin) amplification has been previously reported
for venom immunoassays to improve sensitivity. (Barral-
Netto et al., 1990, 1991; Guo et al., 1993) One study directly
compared the standard peroxidase assay to one with biotin-
avidin amplification and demonstrated the inclusion of the
biotin-avidin amplification made the assay more sensitive.
(Barral-Netto et al., 1990) We have confirmed the significant
benefits of using the biotin-streptavidin amplification which
decreased the LoD from 4 ng/mL, with a standard peroxidise
assay for Australian brown snake, Pseudonaja spp. (O’Leary
et al., 2006), to 0.15 ng/mL here for the taipan venom
assay and 0.3 ng/mL for an assay for rough-scale snake
venom (T. carinatus). (Gan et al., 2009) It is unusual that,
despite the work by Barral-Netto et al and Guo et al. in the
early 1990s, and the widespread use of biotinylation for
enzyme immunoassays, (Strachan et al., 2004) the incor-
poration of biotin-streptavidin amplification into venom
enzyme immunoassays has not become standard. There is
one example of their use in the development of a diagnostic
assay to distinguish major snakes in southern Vietnam.
(Dong et al., 2003).
There are some limitations with this assay including the
potential for cross-reactivity, such that the taipan assay
might detect/measure venom from other snake types as
‘‘taipan’’ venom. Although this is demonstrated in Table 3 it
will only be a problem for low concentration results on the
taipan assay. Distinguishing between different venoms
using enzyme immunoassays is most difficult for low
concentrations where there is only a small separation of the
absorbance versus concentration curves. We have previ-
ously developed an assay that distinguishes between the
closely related genera Notechis, Tropidechis and Hop-
locephalus (tiger
snake
group) venoms. (O’Leary et al.,
20 08; Gan et al., 2009) Although the LoD for this assay
was 0.3 ng/mL for the individual venoms, the limit at which
the three venoms could be distinguished was 2 ng/mL.
Similarly for the taipan assay Table 3 shows that it is
difficult to distinguish between black snake, tiger snake
and rough-scale snake venoms at the LoD. However, above
1 ng/mL much higher concentrations of the other venoms
are required making it easy to differentiate. This shows that
the extent of cross-reactivity declines non-linearly with
increasing concentrations (Table 3). This is most likely
because the ‘‘cross-reactive’’ binding sites of the other
venoms will be rapidly exhausted while taipan venom will
continue to bind anti-taipan antibodies. Any result on the
assay registering less than 1 ng/mL should be repeated with
at least black and tiger snake venoms. Table 3 demonstrates
that there is little cross-reactivity with other Australian
snakes, even close to the LoD. This is most important for
brown snake which is the main snake that can produce
similar early clinical effects to a taipan envenoming in the
same geographical distribution.
An alternative approach to ensure no cross-reactivity
between venom types is that used by Dong et al. (2003) In
this assay the snake venom antibodies are further purified
by immune-affinity chromatography to remove antibodies
that bind to any of the other venoms. This results in highly
specific antibodies for each of the venoms which contain
only antibodies that bind the venom antigens that are
unique to the respective venom. This will result in minimal
cross-reactivity, but will result in an assay that can no longer
be used to quantify the venom concentration, because
antibodies will be missing to major toxin groups (i.e. if the
snakes have similar toxins). This approach worked well for
a diagnostic assay in South-East Asia where the snakes are
very different. However, in Australia where there are simi-
larities between most of the major toxin groups, such as
most of the venoms containing prothrombin activators,
(Isbister et al., 2010b) there may be few venom antigens that
are unique to each snake type. In future assay development
it will be important to determine for each assay if the aim is
to differentiate between snake types (diagnosis/choice of
antivenom) or to quantify venom and use the assay to
determine dose and re-occurrence of venom antigenaemia.
The data on storage cond itions and repeat assays
suggests that it is important for patient samples to be
processed, separated and frozen within hours, a fter wh ich
S. Kulawickrama et al. / Toxicon 55 (2010) 1510–15181516
Page 7
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.
Acknowledgements
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
None.
Funding
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
ID513901.
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  • Source
    • "The concentration of RBBS venom was measured in clinical samples (serum, urine, bite site swab) using a sandwich ELISA. The ELISA was similar to that previously utilised for measuring taipan venom (Churchman et al., 2010; Kulawickrama et al., 2010). RBBS venom specific antibodies were purchased from a commercial supplier (Harry Perkins Institute, Perth, Western Australia). "
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    Full-text · Article · Apr 2016 · Toxicon
  • Source
    • "All samples were immediately centrifuged, and serum aliquoted and frozen at -80°C. Venom was quantified using a sandwich enzyme immunoassay as previously described212223. In brief, rabbit IgG antibodies (provided by University of Rajarata) were bound to microplates as well as conjugated to biotin for the sandwich enzyme immunoassay, detecting with streptavidin-horseradish peroxidase. "
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    Full-text · Article · Feb 2016 · PLoS Neglected Tropical Diseases
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    • " A sandwich EIA was used to measure Russell's viper venom in serum samples and has previously been described.[7, 21, 22] In brief, polyclonal IgG antibodies were raised against Russell's viper (D. russelii) venom in rabbits[23]. Antibodies were bound to microplates as well as being conjugated to biotin for a sandwich EIA with the detecting agent being streptavidin-horseradish peroxidase. All samples were measured in triplicate, and the averaged absorb"
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