1
RATIONALE
Snakebite is one of danger of common accidents in tropical
countries, with over 2.5 million people are bitten by venomous
sankes and about 125,000 people die by venomous snakebites each
year. According to experts about 30,000 people are bitten by snakes
in our country each year. Statistical data of nearly two thousand
patients bitten by venomous snakes at Cho Ray Hospital showed that
species of venomous snakes usually cause the most accidents in the
Southern region of Vietnam is Trimeresurus albolabris (43,3%),
Naja kaouthia (23,8%), Calloselasma rhodostoma (19,4%), Naja
siamensis (10%) and Ophiophagus hannah (1,2%).
In Vietnam, there are two types of commercial antivenom serum
specific with venoms of Trimeresurus albolabris and Naja kaouthia
are being used in clinical. However, the diagnosis of specices of
venomous snakebite to use antivenom serum is depend on clinical
experience and have not a test for detection of snake venoms.
There are many immune techniques is applied to detect snake
venom, in which the enzyme linked – immuno sorbant assay
(ELISA) is often used to develop the test for detection of snake
venom.
From the above theoretical and practical, subject: “Development
of ELISA kit for detection of snake venoms and clinical
application for snake bite diagnosis in Vietnam” was done for
aims:
1. Fabrication of an AB-ELISA kit for detection venoms of four
snake species of Trimeresurus albolabris, Naja kaouthia,
Calloselasma rhodostoma, Ophiophagus hannah and development
2
of an AbE-ELISA kit for detection of venom of two species of
Trimeresurus albolabris and Naja kaouthia in Vietnam.

Vietnam
According to Tran Kien and Nguyen Quoc Thang (1995),
Vietnam has more than hundred species of snakes, including
venomous snakes dozens that distribute both on land and underwater.
In which, some common venomous snakes species cause venomous
snakebite in Vietnam: Trimeresurus albolabris, Naja naja kaouthia,
Calloselasma rhodostoma, Naja naja siamensis and Ophiophagus
hannah
1.1.2. Clinical characteristics of venomous snakebites
The clinical symptoms of snakebite envenomation patients are
varied and plentiful, including local and systemic symptoms.
1.1.3. Diagnosis of venomous snakebite in Vietnam
Diagnosis of venomous snakebite in Vietnam is mainly based on
clinical symptoms and have no test for detection snake venom is
studied and applied to diagnosis of snakebite species identification in
clinical. So development of test for detection of snake venom and
application in diagnosis of venomous snakebite species is necessary
requirements in Vietnam.
1.2. SNAKE ANTIVENOM ANTIBODIES AND
IMMUNOLOGICAL TECHNIQUES FOR DETECTION OF
SNAKE VENOM
1.2.1. Snake antivenom antibodies
1.2.2. Immunological techniques for detection of snake venom
4
13. ELISA
1.3.1. Selection of ELISA test for detection of snake venom
Although many immunological techniques for detection of
venomous snake venom are studied and applied as in section 1.2.2.
However, the most immune technique for detection of venomous
snake venom in manufacture of test is ELISA.

CHAPTER 2
MATERIALS AND METHODS
2.1. SUBJECTS AND STUDY MATERIALS
2.1.1. Subjects of study
- Studies of creating anti-snake venoms monospecific rabbit serum
Twelve healthy gray male rabbits, weighing from 2 to 2,5
kilograms were divided to 4 groups and each group had 3 rabbits.
- Studies of effectiveness assessing the detection of snake venoms of
ELISA kit in clinical
Specimens for detection of snake venoms including 115 whole
bloods, 119 urines and 4 blisters were taken from 122 patients who
was bitten by venomous snake and treated at the Department of
Tropical Diseases in Cho Ray Hospital in Ho Chi Minh city.
2.1.2. Materials
Total venom of four venomous snake species (Trimeresurus
albolabris, Naja kaouthia, Calloselasma rhodostoma and
Ophiophagus hannah) in lyophilized form, the research products of
KCB.04.06.01 project that has accepted in 2009. The chemical,
biological and research equiments are standard analyzed that supply
by reputable manufactures.
6
2.2. METHODOLOGY
2.2.1. Creating antigen and immunizing to produce rabbit anti-
snakevenom antiserum
2.2.1.1. Creating snakevenom antigen
4 snakevenoms antigens were created from 4 lyophilized
snakevenom samples, by method described by Selvanayagam and et
al (1999).
2.2.1.2. Immunizing rabbit with the snakevenom antigen
Rabbits were divided into 4 groups, each group had 3 rabbits: the

- Step 4: add 100 µl avidin – HRP conjugated into each well.
- Step 5: add into each well 100 µl OPD substrate at concentration
0,5 mg/ml that supplemented with 0,006 % H
2
O
2
. Read result after 5
minutes.
Test validation:
- Positive: OD
sample
> X
OD negative control
+ 3SD
- Negative: OD
sample
≤ X
OD negative control
+ 3SD
2.2.6.4. Evaluating the stability and efficiency of AB-ELISA kit in the
laboratory in detecting snakevenom
The stability of AB-ELISA are checked once a month during the
first six months after production.
Efficiency of detection for venomous snake venom of AB-ELISA
in the laboratory are evaluated base on their ability to detect standard
snakevenom sample in PBS-T solution, serum or plasma, whole
blood and urine at different concentrations: 32 ng/ml; 16 ng/ml; 8
ng/ml; 4 ng/ml; 2 ng/ml; 1 ng/ml; 0,5 ng/ml and 0,25 ng/ml.
8
2.2.7. AbE-ELISA kit for detection of venomous snakevenom

2.3. DATA ANALYSIS
Research data is analyzed according to the method of biomedical
statistics
9
Figure 2.1. Research model
10
CHAPTER 3
RESULTS
3.1. AB-ELISA KIT FOR DETECTION OF VENOMS OF 4
SNAKE SPECIES: Trimeresurus albolabris, Naja kaouthia,
Calloselasma rhodostoma and Ophiophagus hannah
3.1.1. Four rabbit antibodies specific to snake species are used in
AB-ELISA kit
Chart 3.4. Result of indirect ELISA for snake species specificity
assessment of anti snake venom antibodies
Comment: Purity rabbit IgG antibodies specific to snake species has
strong reaction to the correlative venom, can be considered the
purity antibodies is snake species specific antibodies.
3.1.2. AB-ELISA kit for detection of venomous snake venom
1050 tests were made with test for sensitivity, stability survey and
clinical trials.
11
3.1.3. The sensitivity and ability to detect venomous snake
venom of AB-ELISA in the laboratory
Table 3.1. The sensitivity of AB-ELISA kit for detection of
venomous snake venom
Samples
Sensitivity (ng/ml)
T.
Albolabris

3,2
0,4
Comment: Results showed that AB-ELISA kit can detect venom of
all kind of samples and the sensitivity of the assay is different depend
on each type of sample and snake venom, but this assay can detect
the venom level at < 3,2 ng/ml.
3.1.4. The stability of AB-ELISA kit
Chart 3.6. Stability of AB-ELISA kit store at 4 C
Comment: The stability of kit are less than 3 months
12
3.2. AbE-ELISA KIT FOR DETECTION OF 2 SNAKE
VENOMS T. ALBOLABRIS AND N. KAOUTHIA
3.2.1. AbE-ELISA kit for detection of venoms of T. Albolabris
and N. Kaouthia
720 tests (240 test bars) were made for sensitivity testing, stability
survey and clinical trials.
3.2.2. Optimal conditions of AbE-ELISA reactions
The optimal conditions for AbE-ELISA reactions obtained as follow:
- Horse antibodies coated in wells: 5,0 µg/ml
- Rabbit IgG antibidies specific for T. Albolabris and N. Kaouthia:
2,0 µg/ml
- HRP conjugated mice antibodies that anti rabbit IgG (antirabbit
IgG – HRP): diluted 1/10,000
- Incubation time of color substrate: 5 minutes
3.2.3. The sensitivity and ability to detect venomous snake venom
of AbE-ELISA kit in the laboratory
Table 3.6. The sensitivity of AbE-ELISA kit for detection of
venomous snake venom
Samples
Sensitivity (ng/ml)

Comment: The OD values of positive control of AbE-ELISA kit are
not difference between period of 3 months and 6 months while the
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kit storage at 4
o
C. The kit is stable for 6 months from date of
manufacture.
3.3. ASSESSING THE EFFICACY FOR DETECTION
VENOMS AND VENOMOUS SNAKE SPECIES
IDENTIFICATION OF ELISA KIT IN CLINICAL
SPECIMENS
3.3.1. Characteristics of study subjects
3.3.2. The assay results for detection of venoms and venomous
snake species identification of AbE-ELISA kit in clinical
specimens
Table 3.11. The assay results of AbE-ELISA kit of specimens:
blood, urine and blister
The assay results
ELISA
Positive
Negative
T. albolabris
N. kaouthia
Blood(n = 115)
23
6
86
Percentage of positive
25,2 %
Urine (n = 119)

45 (62,5 %)
1 (2 %)
46
Negative
27 (37,5 %)
49 (98 %)
76
Sum
72 (100%)
50 (100 %)
122
KAPPA = 0,56
Comment: The positive results of AbE-ELISA assay for detection of
T. Albolabris venom in specimens of patients who had clinial
diagnosis: bitten by T. Albolabris is 62,5%. The efficacy of defind
diagnosis for T. Albolabris species bite by AbE-ELISA assays are
consistent with diagnosis for T. Albolabris species bite in clinical
with KAPPA = 0,56.
Table 3.13. The result of AbE-ELISA assays for detection of T.
Albolabris venoms along with clinical diagnosis to distinguish
envenomization levels by T. Albolabris bite.
Clinical
ELISA assay
Biiten by T. Albolabris
have uncoagulated
Biiten by T. Albolabris
have not uncoagulated
Positive
30 (81,1 %)
15 (42,9 %)

103 (100 %)
122
KAPPA = 0,78
Comment: The positive results of AbE-ELISA assay for detection of
n. kaouthia venom in specimens of patients who had clinial
diagnosis: bitten by common cobra is 68,4%. The efficacy of defind
diagnosis for common cobra species bite by AbE-ELISA assays are
consistent with diagnosis for common cobra species bite in clinical
with KAPPA = 0,78.
Table 3.15. The result distribution of detection for venomous
snake venom along with sample collection time after being bitten
Type of spicemens
and positive rate
ELISA assay
First 3 hours
3-24 hours
After 24
hours
+
-
+
-
+
-
Blood (n=115)
7
13
21
47
1

only 30,9% of blood samples. The AbE-ELISA kit is still possible to
detect venom in urine samples with positive rate was 32,1% after 24
hours being bitten. There is only one case of positive assay result for
blood samples at 28 hours after being bitten as 3,7%.
CHAPTER 4
DISCUSSION
4.1. AB-ELISA KIT FOR DETECTION VENOMS OF 4
SNAKE SPECIES: Trimeresurus albolabris, Naja kaouthia,
Calloselasma rhodostoma and Ophiophagus hannah
4.1.2. AB-ELISA kit for detection venoms of 4 snake species:
Trimeresurus albolabris, Naja kaouthia, Calloselasma rhodostoma
and Ophiophagus hannah
AB-ELISA kit manufactured by us also use 8 wells of a flat
bottom bar as describded in the kit of Cox. The kit is capable for
simultaneous detection of 4 common venomous snake venom in
Vietnam: Trimeresurus albolabris, Naja kaouthia, Calloselasma
rhodostoma and Ophiophagus hannah. The ability of detection for
sanke venom is increased by the use of conjugate avidin-biotin, It is
more hundreds of times than the use of agglutination technique and
detection sensitivity is the same in the use of enzyme labeled
antibidy. This has been demonstrated in the study of Ernesto Amuy
1997,Selvanayagam et al (1999) and Le Van Dong et al (2003).
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4.1.3. The sensitivity of AB-ELISA kit
The sensitivity of AB-ELISA assay for detection of venomous
snake venom are presented in table 3.1 show that the assay can detect
to venom at all kind of specimens include whole blood, plasma,
serum, urine and buffer solution with snake venom detection
sensitivity in the buffer solution from 0,4 to 0,8 ng/ml. The
sensitivity of AB-ELISA for detection of venom in our study results

Zong et al in 2003. The AbE-ELISA kits in this study have
demonstrated the ability detection of venomous snake venom in
biological fluids such as whole blood, plasma and urine.
4.2.4. The cross-reactivity of AbE-ELISA assay with some
venoms of other venomous snake species in Vietnam
By using 4 standard snake venoms of Trimeresurus albolabris,
Naja kaouthia, Calloselasma rhodostoma and Ophiophagus hannah
in difference concentrations to do AbE-ELISA assay. We found that,
there is no false positive reaction of kit for detection venom of
Trimeresurus albolabris and Naja kaouthia with 3 kind of other
standard snake venoms at all levels of standard snake venom < 200
ng/ml that were surveyed.
4.2.5. The stability of AbE-ELISA kit
The stability of AbE-ELISA kit has better than AB-ELISA kit
with 6 months stability time in storage condition at 4
0
C. The AbE-
ELISA kits have better stability because of biologicals use in
fabrication of kits are commercial biologicals, such as horse serum
from IVAC specific for venom of Trimeresurus albolabris and Naja
kaouthia that demonstrated to effective in clinical treatment and
response “directly” between anti snake venom serum and venom in
20
the victems’s body. HRP conjugated mouse antibody that anti rabbit
IgG was suppled by Sigma of America. The rabbit IgG specific for
snake species was obtained in this study in form of total IgG that not
conjugated enzyme or biotin so it has higher stability.
4.3. THE EFFICACY OF DETECTION VENOMS AND
VENOMOUS SNAKE SPECIES IDENTIFICATION OF ELISA
KIT IN CLINICAL SPECIMENS

The positive results of AbE-ELISA assay for detection of n.
kaouthia venom in specimens of patients who had clinial diagnosis:
bitten by common cobra (naja kaouthia and naja siamensis) is 68,4%
(table 3.14). Although the specimens of patients who bitten by
common cobra (naja kaouthia and naja siamensis) is not much, but
the results showed that cross-reactivity occurs in specimens of
pateints who bitten by naja siamensis, while the positive results of
our test is naja kaouthia venom. Thus, ELISA assay for detection of
naja kaouthia venom in this study was only able to distinguish
between patients who bitten by common cobra and patients who
bitten by another neurotoxic snakes.
- The results of AbE-ELISA assay for detection of venomous snake
venom along with sample collection time
The result showed that positive rate of AbE-ELISA assay in the
blood and urine samples was similar to rates of 35% and 39,1%
within first 3 hours after being bitten (table 3.15). Thus, the results of
our study show that venomous snake venom appears in blood and
urine before 3 hours after being bitten. Specifically, we can detect
venomous snake venom in urine after 2 hours of being bitten.
22
In the period of time from 3 to 24 hours after being bitten, the
positive rate of AbE-ELISA assay in urine samples is higher than in
blood samples with respectively rate 51,5% and 30,9% (table 3.15).
This is entirely appropriate because: urinary is the main excretion of
poison out of the body. The venom level of patients who severe
poisoning is tend to higher than patients who without severe
poisoning, the result is excreted much more in urine that make the
positive rate is higher. Beisde of it, urine has reuptake water and
concentrate mechanism so venom level in urine at the time of
sampling of venom is still high enough to positive tests when in the

in Vietnam, with the following specifications.
- AB-ELISA kits are able to detect and distinguigh standard
venom of 4 venomous snake species: Trimeresurus albolabris, Naja
kaouthia, Calloselasma rhodostoma, Ophiophagus hannah that
dissolved into the whole blood, plasma, urine and fluid buffer with
detection thresholds ranging from 0,4 to 3,2 ng/ml depending on the
type of snake venoms and samples. The stability of kit was not
exceed 3 months from the date of manufactured while stored 4
0
C
conditions.
- AbE-ELISA kits are able to detect and distinguigh standard
venom of Trimeresurus albolabris and Naja kaouthia that dissolved
into the whole blood, plasma, urine and fluid buffer with detection
thresholds ranging from 0,5 to 4 ng/ml depending on the type of
snake venoms and samples. The stability of kit was over 6 months
from the date of manufactured while stored 4
0
C conditions.
2. Analysis of 115 blood, 119 urine and 4 blister samples from
122 snakebite patients hospitalized and treated at Cho Ray
24
Hospital showed the efficacy for detection venoms and venomous
snake species identification of ELISA kit in clinical specimens as
follows:
- AbE-ELISA kit was able to detect Trimeresurus albolabris and
Naja kaouthia venoms of all types of clinical specimens include:
whole blood, urine and blister with positive rate respectively: 25,2%;
44,5% và 50 %. The detection rate of T. Albolabris venom of patient
group with clinial diagnosis: bitten by T. Albolabris is 62,5%. This