Snake Venom Detection in Australia

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Snake Venom Detection in Australia Rachel
Jensen IH Business Manager
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Agenda Snake Venom Detection in Australia using
SVDK

• The Problem
• Diagnostic Tool Evolution
• Development
• Principle and Purpose
• Components
• Manufacture and Use
• SVDK Application for Other Snake Species

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The Problem

• Back to the late 1970s
• 5 Monovalent Antivenoms to land snake
  immunotypes, Polyvalent Antivenom and Sea Snake
  Antivenom produced
• MURPHYS LAW - Patients present with
  envenomation, clinicians little experience in
  effective management
• BEST PRACTICE Expose patients to the lowest
  possible dose of foreign antibody

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Australian Snakes

• 5 main land based immunotypes
• Visually difficult to identify
• Snake may not be seen
• Symptoms can be confused
• Which antivenom to treat with?

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Australian Context

• Taipan Immunotype
• Death Adder Immunotype
• Brown Immunotype
• Tiger Immunotype
• Black Immunotype

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Immunoassay Field Use Kit for Snake Venom
Detection

• From 1972, CSL confirmed venom in samples for
  laboratories via radioimmunoassay
• Capillary Tube Kit
• AIM Field use kit capable of detection and rapid
  identification of venom in samples
• 1981 launch of the innovative capillary tube
  enzyme immunoassay
• Identified as a valuable tool but
• difficult to manufacture, time consuming
  assembly, complex, difficult to
  use

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Immunoassay Field Use Kit for Snake Venom
Detection

• AIM Field use kit capable of detection and rapid
  identification of venom in samples but..
• easier to manufacture and use, longer shelf
  life, more sensitive and specific
• Sandwich Enzyme Immunoassay (EIA)
• Qualitative, semi-quantitative tool visual
  identification
• Reformat the kit to overcome the problems
• 3 years in development, team of 9 people
• Patent filed Dec 1988

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Snake Venom Detection Kit Development

• Immunodiagnostic Group
• Development of a new sandwich immunoassay format

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Snake Venom Detection Kit Development

• Comparison of simultaneous vs sequential assay

• Freeze dried capture and conjugate antibodies
  together in the well - usability
• Analysis of components for stability

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Monovalent Antivenom Venoms Neutralised
Tiger Snake Brown Snake Black
Snake Death Adder Taipan

• Tiger Snake (Notechis scutatus)
• Copperhead (Austrelaps superbus)
• Clarence River or Rough Scaled Snake (Tropidechis
  carinatus)
• Blue Bellied or Spotted Black Snake (Pseudechis
  guttatus)
• Pale Headed Snake (Hoplocephalus bitorquatus)
• Red Bellied Black Snake (Pseudechis porphyriacus)
• Broad Headed Snake (Hoplocephalus bungaroides)
• Stephens Banded Snake (Hoplocephalus stephensi)
• Sea Snakes
• Common or Eastern Brown Snake (Pseudonaja
  textilis)
• Dugite (Pseudonaja affinis)
• Gwardar or Western Brown Snake (Pseudonaja
  nuchalis)
• King Brown or Mulga Snake (Pseudechis australis)
• Red Bellied Black Snake (Pseudechis porphyriacus)
• Butlers Mulga Snake (Pseudechis butleri)
• Papuan Black Snake (Pseudechis papuanus)
• Blue Bellied or Spotted Black Snake (Pseudechis
  guttatus)

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Snake Venom Detection Kit SVDK
Principle The antibody causing the most colour
binds the most venom in vitro. This antibody will
most effectively bind and neutralise venom in
vivo.
SVDK Immunotype
Antivenom
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Snake Venom Detection Kit SVDK

• SVDK Purpose
• In vitro detection and immunological
  identification (immunotyping) of snake venom in
  snakebite cases
• Assist clinicians (and veterinarians) to select
  the most efficacious and lowest dose monovalent
  antivenom therapy and give clues to typical
  symptoms caused by species
• Detects common Australian elapid venoms and
  categorises them into one of five medically
  important snake groups Tiger
• Brown
• Black
• Death Adder
• Taipan

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Snake Venom Detection Kit SVDK
3 x Yellow Sample Diluent
Peroxide
Chromogen
Strip Holder
3 x Test Strip
3 x Cotton Swabs
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Freeze Dried Test Strip
1
2
4
3
5
6
7
Tiger
Positive Control
Brown
Death Adder
Black
Taipan
Negative Control
Unused Well
Immunotypes
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Test Strip Manufacture
Step 1 - Coat plate with antibody
Step 2 - Add enzyme conjugated antibody
Step 3 - Freeze dried
Tiger
Brown
Black
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SVDK Method Step 1. Prepare the Test Sample

• ALL samples must be mixed with Yellow Sample
  Diluent
• Bite Site Swab
• Do not wash bite site
• Use moist swab
• Affected Bandage or Cloth Sample
• Cut off a small (1-1.5cm2) section
• Urine Specimen
• Blood Specimen
• May be used, but beware of erroneous results
• Must wash a minimum of 15 times

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SVDK Method Step 2. Add the Test Sample
Add two drops of sample to each of the seven wells
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SVDK Method Step 3. Incubation 10 mins at RT
Reconstitutes components and introduces venom
Tiger
Brown
Black
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SVDK Method Step 4. Washing Step
Washes out unbound venom and conjugate
Tiger
Brown
Black
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SVDK Method Step 5. Add 1 drop Chromogen
Solution
Tetramethlybenzidine (TMB)
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SVDK Method Step 6. Add 1 drop Peroxide Solution
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SVDK Method Step 7. Colour Development
Peroxide TMB Peroxidase Blue colour Watch
for 10 minutes for the first well to show colour
development
Tiger
Brown
Black
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SVDK Method Step 8. Reading Results

• The first well to show colour development is
  diagnostic
• Observe for 10 minutes

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SVDK Method Step 8. Reading Results

• Negative Control
• Normal rabbit IgG as primary/coating antibody
• Rabbit anti-tiger IgG as the conjugate
• Must remain colourless
• Eliminates presence of non-specific reactions
• Positive Control
• Rabbit anti-IgG as primary/coating antibody
• Sheep anti-rabbit IgG as the conjugate
• Must be blue
• Indicates all kit components functioning correctly

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Result Interpretation Example 1

• Sample 1
• Reaction Strength Weak
• Detected Venom Death Adder Immunotype
• Venom sample is from a Common Death Adder
• (Acanthophis antarcticus) from Elliston SA
• If Clinical Envenomation
• Use Death Adder Antivenom

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The SVDK does NOT Speciate Snakes!

• It does two things
• 1. Detects the presence of snake venom in the
  sample under test
• 2. Indicates which monovalent antivenom will most
  effectively neutralise the detected venom

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Applicability to Other Snakes

• Base EIA technology suitable
• Patent expires Dec 09
• Need suitable known pure venom or rabbit IgG
  antibodies
• Is there a need in other regions?

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Support Materials

• CSL actively supports snakebite management
  education in Australia through education
  materials, workshops and presentations.
• CSL also supports PNG through the provision of
  first aid materials translated into local
  languages, donation of SVDKs for research and
  participation in workshops and conferences.

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Support Materials SVDK Technical Information
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Support Materials First Aid Information
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Support Materials Pidgin Motu PNG First Aid
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Support Materials First Aid Information
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Support Materials Snakebite Management Posters
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References

• Theakston RDG, Lloyd-Jones MJ, Reid HA.
  Micro-ELISA for detecting and assaying snake
  venom and venom-antibody. Lancet 1977 2 639-41.
• Sutherland SK. Treatment of snakebite and
  arachnid poisoning. CSL 1978.
• Sutherland SK, Lovering KE. Antivenoms Use and
  adverse reactions over a 12-month period in
  Australia and Papua New Guinea. Med J Aust 1979
  2 671-4.
• Sutherland SK. Rapid venom identification
  availability of kits. Med J Aust 1979 2 602-3.
• Sutherland
• Coulter AR, Harris RD, Sutherland SK. Enzyme
  immunoassay for the rapid clinical identification
  of snake venom. Med J Aust 1980 1 433-5.
• Chandler HM, Hurrell JGR. A new enzyme
  immunoassay system suitable for field use and its
  application in a snake venom detection kit. Clin
  Chem Acta 1982 121 225-30.
• Hurrell JGR, Chandler HM. Capillary enzyme
  immunoassay field kits for the detection of snake
  venom in clinical specimens. Med J Aust 1982 2
  236-7.
• Sutherland SK. Treatment of snakebite in
  Australia and Papua and New Guinea. Aust Family
  Physician April 1990 21-42.
• Cox JC, et al. A novel format for a rapid
  sandwich EIA and its application to the
  identification of snake venoms. J Immunol Methods
  1992 146 213-18.
• Sutherland SK. Antivenom use in Australia.
  Premedication, adverse reactions and the use of
  venom detection kits. Med J Aust 1992 157
  734-739.
• Clancy AM, et al. The effect of species and
  geographical origin on the identification of
  their venom using a commercial assay. Med J Aust
  1997 166 7.
• Williams D, et al. Venomous Bites and Stings in
  Papua New Guinea. AVRU Melbourne 2005.

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SVDK Method Summary

• Test Sample Prepare Test Sample in Yellow Sample
  Diluent (YSD).
• Test Sample Volume Add 2 drops of Test Sample (in
  YSD) to each test well.
• Test Strip Incubation Incubate Test Strip for 10
  minutes, Room Temp (22-24ºC).
• Wash Solution Tap water, purified water, saline
  or buffered saline may be used.
• Washing Test Strip Flick between each wash. Wash
  Test Strip 7 times for bite site and urine, 15
  times for other samples.
• Chromogen Volume Add 1 drop of Chromogen Solution
  to each well.
• Peroxide Volume Add 1 drop of Peroxide Solution
  to each well.
• Results The first test well to show visible blue
  colour within 10 minutes is diagnostic. Refer to
  recommended method for test validation and result
  interpretation.

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Venom Practice Kit VPK

• VPK Purpose
• 6 simulated patient samples from human or
  veterinary cases
• From a range of Australian venomous snakes
  (geographically from across Australia)
• Display a range of reaction strengths
• Designed to be used with the SVDK
• Allows operators to gain experience using the
  SVDK and identifying the snake immunotype
  detected when a positive sample encountered
• Allows staff to be trained in use and
  assessed for competency

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Venom Practice Kit Result Sheet
Snake Venoms Courtesy of Peter Mirtschin at Venom
Supplies www.venomsupplies.com
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Result Interpretation Example 2

• Sample 2
• Reaction Strength Strong
• Detected Venom Tiger Immunotype
• Venom sample is from a Common Tiger Snake
• (Notechis scutatus) from Mt. Gambier SA
• If Clinical Envenomation
• Use Tiger Snake Antivenom

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Result Interpretation Example 3

• Sample 3
• Reaction Strength Weak
• Detected Venom Black Immunotype
• Venom Sample is from a King Brown
• (Pseudechis australis) from inland of Brisbane
  QLD
• If Clinical Envenomation
• Use Black Snake Antivenom

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Result Interpretation Example 4

• Sample 4
• Reaction Strength Strong
• Detected Venom Brown Immunotype
• Venom Sample is from a Eastern Brown Snake
• (Pseudonaja textilis) from Barossa Valley SA
• If Clinical Envenomation
• Use Brown Snake Antivenom

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Result Interpretation Example 5

• Sample 5
• Reaction Strength Strong
• Detected Venom Taipan Immunotype
• Venom Sample is from a Taipan
• (Oxyuranus scutellatus) from Coastal QLD
• If Clinical Envenomation
• Use Taipan Antivenom

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Result Interpretation Example 6

• Sample 6
• Reaction Strength Weak
• Detected Venom Black Immunotype
• Venom Sample is from a Red Bellied Black Snake
  (Pseudechis porphyriacus) from Adelaide SA
• If Clinical Envenomation
• Use Black Snake Antivenom

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Warnings
The colour reaction observation must be performed
as per the instructions

• Colour development must be observed continuously
  for 10 minutes after the addition of Peroxide
  and Chromogen

• The first well to show colour development being
  diagnostic

• Large concentrations of venom in the sample may
  cause rapid colour development and more than one
  blue well at 10 minutes

• Reactions should not be interpreted after 10
  minutes

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Warnings
Users may be confused when more than one well
develops colour

• This is entirely normal and is due to natural
  cross-reactions in Australian snake venoms
• Many Australian snakes have common venom
  components detectable with the SVDK
• Example is the well characterised
  cross-reactivity between King Brown Snake and
  Tiger Snake
• (Refer Product Leaflet or Technical Information
  Booklet for details)