nghiên cứu sản xuất huyết thanh kháng nọc rắn cạp nia đa giá f(ab’)2 từ huyết tương ngựa; đánh giá chất lượng chế phẩm trong phòng thí nghiệm bản tóm tắt tiếng anh

Why choose topics: According to WHO estimates, each year Vietnam needs about 1.7 million units of blood for emergencytreatment services ≈ 2% of the population [40], apart from blood an

INTRODUCTION

1 Why choose topics:

According to WHO estimates, each year Vietnam needs about 1.7 million units of blood for emergencytreatment 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 longcoastline, Vietnam has a very favorable environment for the development of poisonous snakes Mostresidents living and working in the agricultural environment, forests, islands the risk of poisonous snakebites are very high (> 30,000 / year [21]); outside the deaths, costs treatment is expensive: many victimsventilation monthly or tens of liters of blood transfusions and plasma to save lives; records, beginning6/2013 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 ofthousands 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 thespecificity 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 thediagnosis [15] Research antivenom production rates for both multi- poisonous snakes are dangerousaforementioned favorable solution provides for the emergency treatment of patients with snake bite in the

country To increase the safety of antivenom, form F(ab')2 is optimal At the same time, the completeproduction process, standardized products will bring many advantages for the emergency treatment ofpatients in both North and South as well as in the country.

2 Goals topics:

(1) Production research antivenom resistance (BC + BM) polyclonal F(ab')2 Vietnam meet standards(Vietnam Pharmacopoeia)from horse plasma

(2) Evaluate the quality in laboratory

3 The Significance of topics and new contributions:

3.1 For the first time in Vietnam and around the world has produced BC & BM antivenom against two common species BC & BM snake , the most dangerous in the Vietnam and on the world.

3.2 The first time was perfect and standardize all stages of the production process BC&BM antivenom, theend result is a product achieve the National Standards (Vietnam Pharmacopoeia IV, 2009 - latest) Product

3.3 Has produced and purified as successful antivenom F(ab') 2 is as preparations advanced, most effectiveproducts in the form of antivenom; remove the Fc of IgG molecules, to the root of the problemimmunological safety of products using the treatment

3.4 The problem of effective antivenom preparations: has been successfully solved with the synchronoussolution: choosing the right type of snake, quality venom , venom lose virulence but retains antigen; makesure schedules immune adjuvant & antigen dose; follow-up care for horses born best antibody, antibodyplasma hightiter; antibody specific, ensuring sterile blood; F(ab')2 biological activity, purification of F(ab')2has the highest concentration

4 The layout of the thesis:

- Introduction

- Chapter 1: Overview (page 38)

- Chapter 2: Subjects and Methods (18 pages)

- Chapter 3: Research results (31 pages)

- 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

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

1 General safety Satisfactory

2 Sterility no bacteria, fungi

Table 2.2: Criteria to achieve the WHO-Guidelines, 2008[151].

1 General safety Satisfactory

2 Sterility no bacteria, fungi

7 Sodium chlorid 0,85 % - 0,9%

8 Total nitrogen ≤ 100 g/l

2.2.3 Research content:

2.2.3.1 Research BC&BM antivenom produced polyclonal F(ab') 2

- Production of antigens

- Causing susceptible horses, antibody track specific form

- Take blood, plasma collection, transfer payment RBC

- Refined BC&BM polyvalent F (ab ') 2 antivenom, determine the degree of purity of the product

2.2.3.2 Assessing the quality of BC&BM polyvalent F(ab' ) 2:

- Inspection of facilities, assess the safety and efficacy of products

- 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, DavidWarell [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 immuneelectrophoresis with venom

- Collect plasma plasmaferesis method

- Refined BM+BC antivenom F(ab')2 by the method of cutting Fc -globulin by pepsin, precipitated withammonium 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

LD50 , ED50

- After the quality inspection facilities, jarred 5ml/vial sterile and National accreditation requirements

- After the test results National conducted to compare test results to determine the basis of the existingproblems and decide on further processing of batches produced (BC+BM) antivenom Testing facilitiesinclude: general safety test, test pyrogenic, sterility tests, test its effectiveness Testing the efficacy of twosteps: determination of LD50 venom, then determine ED50 of antiveom has produced LD50 calculated by theformula Karber

- National Accreditation: the process of National Institute for vaccines and biologicals, including(8): Generalsafety trials, Sterility testing, Experimental heat release elements, Testing the efficiency (cost efficiency),Total protein concentration, pH, NaCl concentration The concentration of the preservative (Merthiolat)

2.3.5 Time and place of study:

- Study period: 6/2008 - 11/2011

- Location conduct: Research unit antivenom of National Poison control center Bach Mai hospital, NationalInstitute for Control of vaccines and biologicals (Ministry of Health), Center for Empirical research picnicMilitary Medical university, Natioal Institute of hematology and blood transfution , 103 Hospital, Bach MaiHospital and several locations in the Hanoi, Vung Tau

Chapter 3: RESULTS 3.1 PRODUCTION POLYVALENT ANTIVENOM F(ab)2 FOR B CANDIDUS & B MULTICINCTUS SNAKE

3.1.1 Antigen fabrication and evaluation of antigen.

Table 3.1: Results snake selected and venom.

Activity & the

snake venom

(gam)

SLNTB(mg)

- 3 (122) 62 0,5 8,0 60 0,7 11,6

SLNTB: average number venom of a snake / times

Table 3.2: Preliminary evaluation of the level of spending Bungarus snake common in Vietnam

Species

Region

Bungarus fasciatus

Bungarus candidus

Bungarus multicinctu s

Bungarus slowinskii

Bungaru s flaviceps

Note: Not met:(-); Meet but very little number: (+)

Frequent, small amounts (+ +); common, more number: (+ + +)

Table 3.3: The volume of venom antigens and detoxified

venom (mg)

Abnormal Change Increas

e inweight

(g)

fever Loss

hair

weight(g)

Temperature before / afterinjection Antigen(oC) temperatuchange

rehighestStart 1 Hour Hour2 Hour 3

Table 3.6 Results bacterial culture, fungal culture for Antigen

Environment Sabouraud (37oC) Thioglycolate (20-25oC)

3

day

7day

14day

1day

2day

3daynegative negative negative negative negative negative

3.1.2 Results sensitizer horse & monitor the immune response:

Table 3.7: The dose of antigen and adjuvant sensitizer horse.

9 Nom Nom Nom Nom Nom Nom Nom Nom

Table 3.9: Antibody titer after immune horses

Photo 3.4. Traces of the Ag-Ab 1.5% agarose agar (arrow left)

Photo 3.5 Identify specific antigen with BM+BC Ab by immune electrophoresis (right).

3.1.3 Collect blood, separating plasma, erythrocyte volume:

Table 3.10: The volume of blood, plasma, RBC mass is obtained.

Volum

Horse

Blood(lit) Plasma (lit) volume (lit)ErythrocyteHorse 1

3.1.4 Results purified BC+BM polyvalent antivenom F (ab')2:

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.

targetParcel

Plasma (ml) The filtrate is Ab

(ml) Rate filtrate/Plassma(%)

Semi -antivenom(ml)

Dialysis fluidlossing (ml)

Table 3:15: Number of antivenom F (ab')2 was produced

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

Figure 3.6 Compare pictures horse serum protein electrophoresis (1) (left) and BC+BM antivenomF (ab')2finished products (2) (Right):

-3.2 QUALITY ASSESSMENT OF LABO PREPARATION:

3.2.1 Quality control at the facility:

Table 3:18: Safety testing of antivenom in the guinea pig

Num Volum(gam) (ml)AV

inweight(gam)

Losshair

VolumWeek 1 Week 2 Week 3

(ml) Trước Sau 1 h Sau 2 h Sau 3 h (oC)

Table 3:20: Check the level of sterile products

Parcel Sabouraud (37oC) Thioglycolate (20-25oC)

Parcel I negative negative negative negative negative negativePảcel 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)

4 6,66 1,33 1 7 8 12,50

Table 3.22: Determination of effect of antivenom serum

Num Antivenom(ml) BPS venom 40LD50(ml) LD50/mice micedead live total

3.2.2 National test results for quality:

Table 3:23: Results of national tests for quality of product

4.1.1 Production of antigens:

- Selection of snakes, their venom: solid selection, their venom is a very important first step of theproduction process 2 species of snake venom intended has made antivenom-cost good quality, representingthe populations of both species in Vietnam After 6 times conducted collectors get enough of the necessaryvenom 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 ofantigens of different types of volume based on immune dose schedule and is expected to study the horse'sweight Close the vial in each group can build from 0.1 ml to 6 ml, divided into 9 steps to 9 months sensitizerfor horses The number of antigen used to produce enough in research, horses die situation room, study andstorage failures serve antiveom quality control (Table 3.3)

- Quality control antigen: tables 3.4, 3.5, 3.6 showed satisfactory quality of general safety, sterility, pyrogenswhen no sensitizer for experimental animals With the virulent toxin is very strong with sinap nerve - muscle

as venom (BC + BM) , fabricated to induce antigen sensitization if not guaranteed, will cause the animal todie Removes venom toxicity, only retaining antigenicity of toxins are making purposes antigen.Decontamination process with glutheraldehyde, the bacteria have been destroyed in large part, bacteria, fungiwere removed by filtration through a membrane filter at 0.2 micrometers In the fabrication process antigen(venom , venom detoxifying, centrifugal conduct, leaving residue, obtained antigen solution, sterile filtered,extracted in the vial, bottle capping, ), requires no bacteria, eliminate pyrogens have been strictly compliedwith (table 3.5, 3.6)

4.1.2 Process sensitizer horse, monitoring the immune response:

Method 1 month / time period is to ensure safety for horses after immunity (associated ulcers, fatigue,weakness , ) at the same time is the time required for the immune response occurs (according to WHO,needed from 3-8 weeks) Antigen dose causing increased susceptibility, combined with Freund's adjuvantirritating secondary immune response of the horse 's immune system Freund adjuvant used 1 in 2 categoriesincluding: CFA or IFA This is a suspension emulsified antigen (emulsify) in mineral oil, to enhance theimmune response (immunopotentiator) Many authors have used the combined method and CFA antigens toincrease antibody titres showed a remarkably effective Pratanaphon R et al (1997), was mixed land cobravenom antigens Thailand and with different adjuvants such as CFA alone (50% + 50% antigen CFA ), CFA

at a rate of 25% (75% CFA antigens and 25%), vaccine antigens endotoxin + tetanus (tetanus toxoid) vaccineantigen + endotoxin diphtheria (diphtheria toxoid) with different ratios, good results were obtained, highantibody titres increased strongly in the short time compared with the control group only used as antigensand adjuvants bentonite gel

- Monitoring health immune horse after each inject antigens and adjuvants CFA in the first two horses arehorses that fatigue, anorexia, weakness, defeat, located on-site or inactive; injection site average often 2nd