SNAKE BITE PROBLEM AND RELATED RESEARCH QUESTIONS WITH SPECIAL REFERENCE TO SE ASIA

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SNAKE BITE PROBLEM AND RELATED RESEARCH QUESTIONS
WITH SPECIAL REFERENCE TO S-E ASIA

• P. Gopalakrishnakone ,
• Venom and Toxin Research Programme
• YLL School of Medicine
• National University of Singapore

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Research Areas

• Epidemiology.
• Taxonomy.
• Venom collectionstandardization.
• First aid.
• Detection /Diagnostic methods.
• Antisera production/Quality control.
• Local tissue damage.
• Non Traditional methods of treatment.

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Epidemiology

• International statistical classification of
  diseases and related health problems.
• 10th Revision,version for 2000.
• http//www.who.int/classifications/apps/icd/icd10o
  nline/
• (T 63.0 snake venom)---official death
  certification-cause.

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Medically important snakes

• South-east Asia
• Bungarus candidus, B fasciatus, B. multicinctus
• Calloselasma rhodostoma
• Cryptelytrops albolabris,
• C. purpureomaculatus, C. insularis,
• Daboia siamensis
• Naja atra, N. kaouthia, N. philippinensis, N.
  samarensis, N. siamensis, N. sputatrix, N.
  sumatrana
• Ophiophagus hannah

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Snakes of Medical Importance in India
1)Bungarus Caeruleus. 2)Daboia Russelii. 3)Echis
carinatus. 4)Naja naja
1)Hypnale hypnale. 2)Naja Kaouthia.
1)Ophiophagus hannah. 2)Bungarus
fasiatus,niger,walli,sindanus 3)Naja
oxiana. 4)Cryptelytrops(Trimerusurus)albolabris,
purpureomaculatus.
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Taxonomy Identification of snakes.

• Naja species.
• Echis species.
• Daboia species.
• Cryptelytrops(Trimeresurus)species.
• COLOUR VARIATIONS.

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Snake venom collection ,storage
standardization.

• I ndia----Irula soc of Tamil Nadu.
• Haffkine Institute,Bombay
• King Institute,Chennai.
• Bangaladesh.
• Cambodia.
• Nepal. Antisera from India
• Srilanka

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• Thailand---Thai Red cross.
• Japan -----2 centres.
• Vietnam -2 centres.
• China------2 centres.
• Malaysia----Antisera from CSL India

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FIRST AID

• Cleaning of wound.
• Cutting of wound.
• Applying pressure or suction.
• PRESSURE IMMOBLIZATION METHOD.

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DIAGNOSTIC METHODS

• Immunodiffusion method.
• ELISA.
• OIA
• PROTEIN ARRAY.
• QUANTUM DOTS

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PAST (before 2002)Immunoassays for snake venom
and toxin detection

• Immunoprecipitation
• Immunodiffusion
• Radioimmunoassay
• Fluorescent immunoassay
• ELISA
• Ion-sensitive field effect transistor
  (ISFET)-based immunosensor

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RECENTLY(After 2002)

• ELISA, optical immunoassay (OIA), and microarray
  immunoassay for detection of venoms and toxins
  are being used.

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Protocol to prepare snake species-specific
antibodies for diagnosis the of snake species
which caused the bite

• Simple Using conventional affinity
  chromatography techniques
• Cheap Applicable to polyclonal antibody all
  chemical and reagents used are standard ones
• Effectively removes cross-reacting antibody
  molecules

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Preparation of species-specific Ab (SSAb)
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Preparation of SSAb (cont.)
Removal of cross-reacting antibody molecules by
immuno-adsorption with heterologous venom columns
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Efficiency of affinity chromatography protocol in
preparation of snake species-specific antibody
(evaluated by ELISA)
Before purification
Species-specific antibodies
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Efficiency of affinity chromatography protocol in
preparation of snake species-specific antibody
(evaluated by blotting)
Dot blot
Western blot
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ELISA test kit for snakebite diagnosis in South
Vietnam
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Application of ELISA in clinics
Snake venom detection in human samples by
species identification kit
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Application of ELISA in clinics (cont.)
Snakebite diagnosis with snake species
identification kit
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Application of ELISA in clinics (cont.)
Venom detected in different types of samples
Venom kinetics in snakebite patients serum
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Application of ELISA in clinics (cont.)
Overall venom quantitation in samples from
snakebite patients in South Vietnam

• Theakston and Wyatt (1985) 7.5 ng/ml for venom
  of young puff adder a day after the bite
• Viravan et al (1986) 361 ng/ml in 20 cases of
  severe systemic envenomation of N. kaouthia venom
• Ho et al (1986a) 0-600 ng/ml in the plasma of
  patients bitten by C. rhodostoma in Malaysia
• De, (1996) 0-479 ng/ml (mean value of 200 ng/
  ml) in snakebite patients in India
• Hung et al. (2003) 0 - 1,270 ng/ml in blood of
  patients bitten by N. atra in Taiwan

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Optical immunoassay (OIA)

• OIA correlated well with ELISA
• Animal model has been done
• The test works well with clinical samples (83
  patients)

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Semi-quantitation of venoms by OIA
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Simultaneous detection of snake species and venom
semi-quantitation
(A)
(A) Arrangement of the OIA chip for simultaneous
detection of snake species and venom
semi-quantitation (B) Chip 1 positive to all
four venoms Chip 2 T. albolabris venom at 10
ng/ml Chip 3 C. rhodostoma venom at 50 ng/ml
Chip 4 negative (BSA) Chip 5 N. kaouthia venom
at 10 ng/ml Chip 6 O. hannah venom at 20
ng/ml (C) Graphic presentation of N. kaouthia
venom positive
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VENOM/ANTIVENOM MICROARRAYS

• Application of DNA facilities in antibody and
  antigen microarrays

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Solid supports
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Evaluation of solid supports for antibody
immobilization
Parameters of antibody microarray on different
solid supports ()
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Evaluation of solid supports for venom
immobilization
Parameters of venom microarray on different solid
supports ()
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Applications of venom microarray
Cross-reaction between antisera and heterologous
venoms by venom microarray
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Applications of venom microarray (cont.)
Cross-reactivity of venom antiserum to
heterologous venoms ()
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• Cross-reactions were seen between antivenoms and
  venoms of snakes of the same family as well as
  snakes of different family from different
  geographic regions
• Higher cross-reactivity was seen between
  antivenoms and venoms of snakes of the same
  family

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• Highest fluorescent intensities were recorded at
  the spots where the monovalent antisera reacted
  with the homologous venoms
• Venom microarray could be used for retrospective
  study of the epidemiology of snakebite and the
  responsible snake species

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Antivenom microarray for snake venom detection
Snake species identification
Simultaneous detection
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• Snake species of the venom analyte are indicated
  by position and the colour of the positive spots
• The antivenom microarray can distinguish venoms
  of the four common venomous snakes of South
  Vietnam as shown in panels C, D, E, and F
• Internal standards can be incorporated in the
  microarray for simultaneous detection of snake
  species and venom quantitation

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FUTURE TRENDS

• Amperometric immunosensor
• Microbead assays
• Quantum dots

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Amperometric immunosensor
Protein toxin/electrochemical activator bilayer
detection platform Capture antibody was
immobilized on thiol-coated gold electrode. The
test sample was applied on the electrode and the
protein analyte was captured by immobilized
antibody. Biotinylated detecting antibody was
then added followed by avidin-HRP conjugate and
redox polymer. The presence of the analyte was
indicated by the deoxidization current of H2O2
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Amperometric responses of sample toxin at
different concentrations
(I) (a) 10 ng/mL (b) 5.0 ng/mL (c) 1.0 ng/mL (d)
0.1 ng/mL (e) Negative control. The higher the
toxin concentrations, the higher the
deoxidization current recorded (II) Calibration
plot of sample toxin at different concentrations
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Microbead immunoassay for snake venom detection
Polystyrene microbeads coated with antibody for
venom/toxin detection
NEGATIVE POSITIVE
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Microbead immunoassay for the detection of N.
kaouthia venom
Negative control
Positive (0.5 ?g/ml) as seen by fluorescent
microscope
Positive (0.5 ?g/ml) as seen by confocal
microscope
Fluorescent intensities of the beads are
proportional to the venom concentration

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Advantages and potential applications of
microbeads

• More protein can be immobilized on the bead than
  planar surface
• The reaction takes place in liquid-liquid phases
  so as more sensitive than liquid-solid ones
• Beads can be arrayed in an microarray formats
• Beads of diffeent colours and be used for
  multianalyte detection

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Quantum dots (QDs)

• What are quantum dots?
• Nanocrystalline particles
• CdSe and ZnS
• Particles 2-10 nm in size (equal to size of
  protein molecules)

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Different sizes different colours
(A) Nanocrystals absorb light and then re-emit
the light in a different colours the size of the
nanocrystal determines the colour of the QD (B)
Six different quantum dot solutions are shown
excited with the same long-wave UV lamp
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QD bound toxin can be seen visually or
fluorescent intensity can be measured with a
machine for quantiattion purpose
Negative Positive 1/200
1/100
Reaction wells seen under UV light. Positive
samples are lighting up
Dose-response curve in sandwich QD immunoassay to
detect beta-bungarotoxin
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Potential applications of QDs

• Different antibodies can be tagged with QDs of
  different colours for multi-toxin/venom detection
• QDs can be used as fluorescent tags for protein
  trafficking and protein-protein interaction
  studies (in the case of toxin, that is the mode
  of action of toxin)

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ANTI SNAKE VENOM. 1

• ProductionQC of venom,immunization
  schedule,Adjuvents.
• Purification methods.
• Neutralization potency.
• Pharmacokinetics.
• Pharmacodynamics.

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Anti snake venom 2

• Which animal?Horse,Sheep,Chicken,Camel
• Mono specific or Poly Specific?
• Cross neutrlization,Para specificity?
• Allergic reactions??

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LOCAL TISSUE DAMAGE.

• Anti venom.
• Antidotes.
• Antichelating agents.
• Activated charcoal.
• Stem cells.

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Local tissue damage(ctd)

• Tannic acid.
• Tumerin.
• Aristolochic acid.
• Oleanolic acid.
• Chelating agents.
• Hyaluronidase Inhibitors

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Suggestions

• Task force for States/National /Global.
• Clinical networksstates,national,data banks.
• Reference centres-QC of venoms.
• Analysis of venoms.
• Antisera research.
• Educational materialsprint,electronic media