1
INTRODUCTION
1. Why choose topics:
According to WHO estimates, each year Vietnam needs about 1.7 million units of blood for emergency
treatment services (≈ 2% of the population) [40], apart from blood and blood products, plasma products, and
to included serum preparations such as gamma globulin, globulin anti-HBs, anti-T-lymphocyte globulin,
antivenom [141],[147],[152] in which antivenom is very important, especially in the treatment of
hemostatic coagulation disorders due to snake venom poisoning (Vipridae).
As a tropical country, with three-quarters of mountain forests and agricultural lands, over 3000 km long
coastline, Vietnam has a very favorable environment for the development of poisonous snakes. Most
residents living and working in the agricultural environment, forests, islands the risk of poisonous snake
bites are very high (> 30,000 / year [21]); outside the deaths, costs treatment is expensive: many victims
ventilation monthly or tens of liters of blood transfusions and plasma to save lives; records, beginning 6/2013
Bach Mai hospital was ≈ 46 liters of blood transfusion and preparations for saving a patient [174].
To reduce mortality, reduce the number of blood and plasma used to treat poisonous snake bites,
Ministry of Health was interested in directing the research, production applications snake venom serum
antibodies[6],[8]. To date, there have been some very successful research, contributing to save the lives of
2
thousands of patients, significantly reduced blood flow and preparations to use [6],[21]
However, after many years of effort, to date we are still severely lacking in many types of antivenom;
almost like we have only two types of land for cobra and viper bamboo [8],[165],[166],[171]. Due to the
specificity of snake venom antigens geographically, each country must make antivenom for yourself (WHO)
[141],[20],[6]. To meet emergency needs, treating poisonous snake bites, so we need to promote research,
production xuatantivenom; HTKNR especially for dangerous venomous snakes, common, in that species,
leading to nia mention solid waistband, accounting for 35.8% of their poisonous copperhead snakes Elapidae
in Vietnam, the poisonous snakes have extremely strong toxicity, causing very high mortality (> 80% if it is
not an emergency timely treatment) [14].
Actual treatment in Vietnam so far show that have common Bungarus, mainly Bungrarus candidus and
Bungarus multicinctus. These two snake -shaped "piece of black, white songs" are very similar, it is difficult
to distinguish glance. Two different species of snake venom toxicity and the clinical manifestations,
diagnosis is very easy to confuse, antivenom monospecific not work and there is no VDK to confirm the

- Chapter 4: Discussion (37 pages).
- Conclusions
The thesis is: 128 pages, 29 tables, 4 charts and diagrams, 63 photographs and drawings (48 photos annex),
175 references (40 Vietnamese, English 114, 21 sites), 4 appendices.
Chapter 1: OVERVIEW
1.1. Blood and plasma
1.2. The preparations of blood and plasma products
5
1.3. Extraction of plasma components
1.4. Serum anti-snake venom
1.5. By BC, BM accident and production BC+BM antivenom.
Chapter 2: SUBJECTS & METHODS
2.1. Object & study material:
Studies on laboratory animals: snake, horses, rabbits, guinea pigs, white mice. Materials : venom of BC
& BM: 3.1 g.
2.2. Research Methodology:
2.2.1. Study design: laboratory and experiments on animals.
2.2.2. Product quality standards to be achieved:
Table 2.1: National Standards, Vietnam Pharmacopoeia IV, 2009
Num The target Standard to be achieved
1 General safety Satisfactory
2 Sterility no bacteria, fungi
3 Pyrogen no pyrogen
6
4 Antibody titer / vial > 100 LD50/lọ (5ml)
5 pH 6 - 7
6 Merthiolat ≤ 0,01%
7 Sodium chlorid 0,85 % - 0,9%
8 Total nitrogen ≤ 15 %
Table 2.2: Criteria to achieve the WHO-Guidelines, 2008[151].

- Inspection of National, safety assessment, effect, physical and chemical characteristics.
- Preliminary production costing, evaluation of economic efficiency and social.
2.2.4 . The method of research techniques :
- Selection of snakes, venom , venom preserved by the method of Tran Kien, Trinh Xuan Kiem, David
Warell [21],[24].
- Production of antigens and quality evaluation method of Trinh Xuan Kiem and recommended by WHO &
Vietnam Pharmacopoeia.
- Causing susceptible horses, track -specific antibody formation in Ouchterlony tests and serum immune
electrophoresis with venom .
- Collect plasma plasmaferesis method .
- Refined BM+BC antivenom F(ab')
2
by the method of cutting Fc
γ
-globulin by pepsin, precipitated with
ammonium sulphate segments , dialysis with distilled water, filtered Seize. Inspection of facilities: evaluation
of general safety test, heat release factor, bacterial culture, fungal culture, and titer determination LD
50
,
ED
50
.
- After the quality inspection facilities, jarred 5ml/vial sterile and National accreditation requirements .
9
- After the test results National conducted to compare test results to determine the basis of the existing
problems and decide on further processing of batches produced (BC+BM) antivenom. Testing facilities
include: general safety test, test pyrogenic, sterility tests, test its effectiveness. Testing the efficacy of two
steps: determination of LD
50
venom, then determine ED

(mg)
activity 1(114) 84 0,5 5,9 30 0,3 10
- 2 (185)
130 0,6 4,6 55 0,5 9,0
- 3 (122)
62 0,5 8,0 60 0,7 11,6
Total (421) 276 1,6 5,79 145 1,5 10,3
SLNTB: average number venom of a snake / times
Table 3.2: Preliminary evaluation of the level of spending Bungarus snake common in Vietnam
Species Bungarus Bungarus Bungarus Bungarus Bungarus
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Region
fasciatus candidus
multicinctu
s
slowinskii flaviceps
North VN (++) (-) (+++) (-) (-)
South VN (+) (+++) (-) (-) (-)
Note: Not met:(-); Meet but very little number: (+)
Frequent, small amounts (+ +); common, more number: (+ + +)
Table 3.3: The volume of venom antigens and detoxified
Category
Antigen
Antigen volum /vial (ml)
0,1 0,3 0,5 1 2 3 4 5 6
12 12 12 22 12 12 12 12 12
12
Number vial
Number mg
venom/vial ≈

(kg)
Vol. Ag
(ml)
venom
(mg)
Temperature before / after
injection Antigen(
o
C)
change
temperature
highest
Start 1 Hour 2 Hour 3 Hour
1 2,1 2,1 23,1 38,5 38,9 39,0 38,5 0,5
o
C
2
2, 3 2,3 25,3 39,1 39,8 39,2 39,1 0,7
o
C
3
2,2 2,2 24,2 39,3 39,5 39,8 39,3 0,5
o
C

Table 3.6. Results bacterial culture, fungal culture for Antigen
Environment Sabouraud (37
o
C) Thioglycolate (20-25
o

in place
The whole body
in
place
Mov
respirati
on
digestion
Mov
respiration digestion
1
weak
Nom Not eat ulcers
weak
Nom Not eat ulcer
2
weak
Nom Not eat ulcers
weak
Nom Not eat ulcer
3
weak
Nom Eat les ulcers
weak
Nom Eat les ulcer
4 Nom Nom Nom Nom Nom Nom Nom Nom
5 Nom Nom Nom Nom Nom Nom Nom Nom
6 Nom Nom Nom Nom Nom Nom Nom Nom
7 Nom Nom Nom Nom Nom Nom Nom Nom
8 Nom Nom Nom Nom Nom Nom Nom Nom

(TP)
Time 1 2,3 1,4 0,9
Time 2 5,5 3,3 2,2
Total 16,5 10,0 6,5
Table 3:11: The amount of blood taken and the horse's reactions
Blood volum
(lit)
Expression of horses
Normal Light shock Shock
Horse 1
Time 1 3,6
X X
Time 2 5,1
X X
Horse 2
Time 1 2,3
X
Time 2 5,5
X X X
3.1.4. Results purified BC+BM polyvalent antivenom F (ab')2:
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3.1.4.1. Cut Fc, precipitated protein was not antivenom with 14% ammonium sulphate, filtered precipitate more
F (ab ')2:
Table 3.12: The volume of the filtrate with F (ab ') 2 obtained.
target
Parcel
Plasma (ml) The filtrate is Ab
(ml)
Rate filtrate/
Plassma(%)

510
485
25 (5,2%)
Total
700
650
50 (7,7%)
Table 3:15: Number of antivenom F (ab')2 was produced
Product Unit Volum (ml) Number (vial)
Semi – BC+BM antivenom Bottle 500 ml 650 2
Polyvalent antivenom F(ab’)2 Vial 5 ml 650 130
3.1.4.3. Determination of purity of Polyvalent antivenom F(ab’)2
Table 3:17: Quantification of protein, albumin, globulin, IgG, IgM, ratio A/G horse serum and polyvalen
antivenom F (ab')2 of BM&BC
Test Horse antivenom F (ab')2 of BM&BC
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serum Number Rate
Protein (g/l) 87,1 49,8 100 %
Albumin (g/l) 28,7 1,4 2,8 %
Globulin (g/l) 58,4 48,4
100% 97,2 %
IgG (mg/dl) 865 203
2,141 g/l
≈ 4,4%
IgM (mg/dl) 67,8 8,6
IgA (mg/dl) 6,3 2,5
Tỷ lệ A/G 0,49 0,03

Figure 3.6. Compare pictures horse serum protein electrophoresis (1) (left) and BC+BM antivenomF (ab')2-
21

(ml) (
o
C)Trước Sau 1 h Sau 2 h Sau 3 h
1 2,8 2,8 39,3 39,6 39,8 39,3 + 0,5
2 2,8 2,8 39,4 39,7 39,8 39,4 + 0,4
3 3,0 3,0 39,6 39,8 39,9 39,6 + 0,3
Table 3:20: Check the level of sterile products
Parcel
Sabouraud (37
o
C) Thioglycolate (20-25
o
C)
Day 3 Day 7 Day 14 Day 3 Day 5 Day 7
Parcel I
negative negative negative negative negative negative
Pảcel II
negative negative negative negative negative negative
Table 3:21: Determination of lethal dose 50% of BC+BM venom
Number
Venom
(µg/ml)
venom/
Swiss mice
(µg)
Swiss mice
rate
(%)
dead live total
23

4 Titer test 267,5 LD
50
/lọ
5 NaCl 0,87%
6 pH 7.012
7 Protein 7,43 mg/ml
8 Merthiolate content 0 g%
Chapter 4: DISCUSSION
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4.1. PRODUCTION RESEARCH ANTIVENOM F (ab')2 FOR B.CANDIDUS & B.MULTICINCTUS
MEET STANDARDS:
4.1.1. Production of antigens:
- Selection of snakes, their venom: solid selection, their venom is a very important first step of the
production process. 2 species of snake venom intended has made antivenom-cost good quality, representing
the populations of both species in Vietnam. After 6 times conducted collectors get enough of the necessary
venom 2 species north and south. BM&BC sting less, difficult to obtain and very dangerous (Table 3.1).
- Identify common types of solid bay in Vietnam: Vietnam has many compartments snakes "black song,
blanks", easily confused with each other (Table 3.2) is B. candidus, B. multicinctus and Red River snake (B.
slowinskii), in which is 2 common species, have the most number of natural, necessary for producing
antivenom treatment, other less common species.
- Results fabrication BC+BM venom antigen polyclonal, attenuated:
Using 3 g of pure venom of the 2 species, are made 272.8 ml of antigen attenuated. Dividing the number of
antigens of different types of volume based on immune dose schedule and is expected to study the horse's
weight. Close the vial in each group can build from 0.1 ml to 6 ml, divided into 9 steps to 9 months sensitizer
for horses. The number of antigen used to produce enough in research, horses die situation room, study and