1 INTRODUCTION 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 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 diagnosis [15] Research antivenom production rates for both multi- poisonous snakes are dangerous aforementioned 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 complete production process, standardized products will bring many advantages for the emergency treatment of patients in both North and South as well as in the country Goals topics: (1) Production research antivenom resistance (BC + BM) polyclonal F(ab') Vietnam meet standards (Vietnam Pharmacopoeia) from horse plasma (2) Evaluate the quality in laboratory 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, the end result is a product achieve the National Standards (Vietnam Pharmacopoeia IV, 2009 - latest) Product was 9/10 basic indicators of quality of WHO 3.3 Has produced and purified as successful antivenom F(ab') is as preparations advanced, most effective products in the form of antivenom; remove the Fc of IgG molecules, to the root of the problem immunological safety of products using the treatment 3.4 The problem of effective antivenom preparations: has been successfully solved with the synchronous solution: choosing the right type of snake, quality venom , venom lose virulence but retains antigen; make sure schedules immune adjuvant & antigen dose; follow-up care for horses born best antibody, antibody plasma high titer; antibody specific, ensuring sterile blood; F(ab')2 biological activity, purification of F(ab')2 has the highest concentration 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, charts and diagrams, 63 photographs and drawings (48 photos annex), 175 references (40 Vietnamese, English 114, 21 sites), 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 Num The target Standard to be achieved General safety Satisfactory Sterility no bacteria, fungi Pyrogen no pyrogen Antibody titer / vial > 100 LD50/lọ (5ml) pH 6-7 Merthiolat ≤ 0,01% Sodium chlorid 0,85 % - 0,9% Total nitrogen ≤ 15 % Table 2.2: Criteria to achieve the WHO-Guidelines, 2008[151] Num The target Standard to be achieved General safety Satisfactory Sterility no bacteria, fungi Pyrogen no pyrogen Antibody titer / vial Registration standards pH 6–7 Matter content Phenol < 2,5 g/l preservation Cresols < 3,5 g/l Sodium chlorid 0,85 % - 0,9% Total nitrogen ≤ 100 g/l Albumin ≤1% 10 Globulin > 90% 2.2.3 Research content: 2.2.3.1 Research BC&BM antivenom produced polyclonal F(ab') - Production of antigens - Causing susceptible horses, antibody track specific form - Take blood, plasma collection, transfer payment RBC - Refined BC&BM polyvalent F (ab ') 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, 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') 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 , 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 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 LD50 venom, then determine ED50 of antiveom has produced LD50 calculated by the formula Karber - National Accreditation: the process of National Institute for vaccines and biologicals, including(8): General safety 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, National Institute for Control of vaccines and biologicals (Ministry of Health), Center for Empirical research picnic Military Medical university, Natioal Institute of hematology and blood transfution , 103 Hospital, Bach Mai Hospital and several locations in the Hanoi, Vung Tau Chapter 3: RESULTS 10 3.1 PRODUCTION POLYVALENT ANTIVENOM F(ab)2 MULTICINCTUS SNAKE FOR B CANDIDUS & B 3.1.1 Antigen fabrication and evaluation of antigen Table 3.1: Results snake selected and venom Bungarus multicinctus Activity & the snake venom snake Bungarus candidus venom SLNTB (gam) snake (mg) venom SLNTB (gam) (mg) activity 1(114) 84 0,5 5,9 30 0,3 10 - (185) 130 0,6 4,6 55 0,5 9,0 - (122) 62 0,5 8,0 60 0,7 11,6 276 1,6 5,79 145 1,5 10,3 Total (421) 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 17 Volum Blood (lit) Horse Plasma (lit) Erythrocyte volume (lit) Horse Time 3,6 2,2 1,4 (VP) Time 5,1 3,1 2,0 Horse Time 2,3 1,4 0,9 (TP) Time 5,5 3,3 2,2 16,5 10,0 6,5 Total Table 3:11: The amount of blood taken and the horse's reactions Expression of horses Blood volum (lit) Normal Light shock 3,6 X X 5,1 X X Time 2,3 X Time Horse Time Time Horse 5,5 X Shock X 3.1.4 Results purified BC+BM polyvalent antivenom F (ab')2: X 18 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 ') obtained target Plasma (ml) The filtrate is Ab (ml) Parcel Parcel Parcel Total : 3600 6400 10.000 2900 5140 8040 Rate filtrate/ Plassma(%) 80,6% 80,3% 80,4% 3.1.4.2 Precipitated F (ab ') with 22% ammonium sulphate, filtration Whatman filter paper obtained by precipitation, dialysis, and sterile filtration generated antivenom Table 3:13: Precipitation amount of F (ab') obtained target Parcel The filtrate is F(ab’)2 precipitation has F (ab ') (ml) obtained (gam) Parcel 2900 210 Parcel 5140 395 Total 8040 605 19 Table 3.14 Dialysis fluid & antivenom semi product Chỉ số NC Dialysis fluid (ml) Semi -antivenom (ml) Dialysis fluid Parcel 190 165 25 (13,1%) Parcel 510 485 25 (5,2%) 700 650 50 (7,7%) Parcel Total lossing (ml) 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 Polyvalent antivenom F(ab’)2 Vial 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 20 Protein (g/l) Albumin (g/l) Globulin (g/l) IgG (mg/dl) IgM (mg/dl) IgA (mg/dl) Tỷ lệ A/G serum 87,1 28,7 58,4 865 67,8 6,3 0,49 Number 49,8 1,4 48,4 203 8,6 2,5 2,141 g/l ≈ 4,4% Rate 100 % 2,8 % 100% 97,2 % 0,03 Figure 3.6 Compare pictures horse serum protein electrophoresis (1) (left) and BC+BM antivenomF (ab')2- 21 finished 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 Guinea pig Num Volum AV (ml) Loss (gam) hair Increase in weight (gam) Volum 310 3,1 Week 320 Week 325 Week 340 +30 330 3,3 343 350 355 +25 350 3,5 358 363 370 +20 Table 3:19: Identify pyrogenic in semi-finished products Volum (kg) Num Vol antivenom Temperature before / after injection antivenom (oC) Nhiệt độ chênh lệch 22 (ml) Trước Sau h Sau h Sau h (oC) 2,8 2,8 39,3 39,6 39,8 39,3 + 0,5 2,8 2,8 39,4 39,7 39,8 39,4 + 0,4 3,0 3,0 39,6 39,8 39,9 39,6 + 0,3 Table 3:20: Check the level of sterile products Sabouraud (37oC) Parcel Thioglycolate (20-25oC) Parcel I Day negative Day negative Day 14 negative Day negative Day negative Day 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 dead live rate total (%) 23 22,50 4,5 8 100,00 15,00 3,00 87,50 10,00 2,00 4 50,00 6,66 1,33 12,50 4,44 0,88 8 0,00 0 8 0,00 Table 3.22: Determination of effect of antivenom serum mice Num Antivenom (ml) BPS venom 40 LD50(ml) LD50/ mice dead live total 0.000 2.000 0.000 0 8 0.000 1.000 1.000 8 0.060 0.940 1.000 0.070 0.930 1.000 4 24 0.080 0.920 1.000 3.2.2 National test results for quality: Table 3:23: Results of national tests for quality of product Target Testing Result Safety test Satisfactory Pyrogen test Satisfactory Sterile test Satisfactory Titer test 267,5 LD50/lọ NaCl 0,87% pH 7.012 Protein 7,43 mg/ml Merthiolate content g% Chapter 4: DISCUSSION 25 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 species of snake venom intended has made antivenom-cost good quality, representing the populations of both species in Vietnam After times conducted collectors get enough of the necessary venom 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 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 g of pure venom of the 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 ml, divided into steps to months sensitizer for horses The number of antigen used to produce enough in research, horses die situation room, study and 26 storage 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, pyrogens when 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 to die 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, fungi were 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 complied with (table 3.5, 3.6) 4.1.2 Process sensitizer horse, monitoring the immune response: - Schedule horses immune, antigen dose and adjuvant: Method 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 adjuvant 27 irritating secondary immune response of the horse 's immune system Freund adjuvant used in categories including: CFA or IFA This is a suspension emulsified antigen (emulsify) in mineral oil, to enhance the immune response (immunopotentiator) Many authors have used the combined method and CFA antigens to increase antibody titres showed a remarkably effective Pratanaphon R et al (1997), was mixed land cobra venom 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) vaccine antigen + endotoxin diphtheria (diphtheria toxoid) with different ratios, good results were obtained, high antibody titres increased strongly in the short time compared with the control group only used as antigens and adjuvants bentonite gel - Monitoring health immune horse after each inject antigens and adjuvants CFA in the first two horses are horses that fatigue, anorexia, weakness, defeat, located on-site or inactive; injection site average often 2nd and 3rd injection repeated after month, saw horses stop eating or eat less , injection site ulcers appeared, 37 cm in diameter These injections with IFA adjuvants, injection site ulcers not happen; usual manifestations, horses still traveling, eating good, healthy posture The phenomenon occurs after skin ulcers in horses immune adjuvant CFA 2.3 second time but not the cause ulcers in susceptible adjuvant IFA (table 3.8) (death due to BCG CFA) According to WHO, only once CFA injection and injection at a limited number of large 28 antigen + adjuvant, but the team has not complied with this horse still alive and immune response occurs normally, but care harder; did not prove effective immune response is higher than WHO guidelines - Monitoring antibody titer after immunity: After times immunity, antibody titres specific for antigens venom rose to 1/2048 second in both horse and stable after immune 8th and 9th Thus, blood can get more soon after visits immunity by snake venom antigens and adjuvants Results Table 3.9 also shows that, with two strong horses, and the equivalent weight is raised in different locations nnhau, the same course immune to the same doses, may result in different antibody titres together This suggests: care regimen and nutrition plays an important role in the immune response of the horse - Determination of antibody specificity horses with venom antigens: Using Ouchterlony test after test sensitizer, see the stain precipitate appearing Ag-Ab agar, horse blood was demonstrated in antibody formation Serum mixed with different concentrations to examine the degree of antibody formation When producing polyclonal antivenom F (ab')2, as the immune cell phone with venom antigens, appear very clearly see traces precipitated Ag-Ab agar, this effect confirmed antibody specificity is very good (Fig 3.4, 3.5) 4.1.3 Taking blood, separated plasma, transfer payment: 29 - Sterile sampling , blood was not crowded, not broken erythrocytes, horses are taken to ensure safe blood , blood plasma separation immediately took complete return to television for horse erythrocyte volume(Table3.10) - When the note receivable plasma and blood taken by horse health , blood and get enough to produce a normal horse Results Table 3:11 : when less than 3.5 liters of blood , the two horses are not showing symptoms When taking 3.6 - > 5.4 liters of blood, depending on the horse , different tolerance, the degree of response to different blood sampling During blood volume obtained above, when abnormal , blood stops handling time, horses are safe, not dangerous The level of blood drawn more than 5.5 liters/horse, if not pay attention happens to horses by unsafe blood loss shock 4.1.4 Purified polyclonal antivenom F(ab')2 high quality: - Cut Fc, not precipitated protein antibody precipitate filtered, the filtrate collection contains more F(ab') 2: Molecule IgG, Fc fragment intact there, that's cause unwanted reactions in clinical especially anaphylactic shock Fc receptor-bearing cells attached mastocyte, basophilic leukemia is an intermediate storage of the chemical histamine, serotonin, bradykinin cause anaphylaxis, the most dangerous type of reaction in antisera therapy We used to remove Fc Pepsin, keep the F(ab')2 The rate of capture rich plasma F(ab')2 with 30 refined is 80.4% (Table3:12) Thus, ~ 20% of the precipitation of plasma containing no antibodies and F(ab')2 It is first necessary to purify the product Ammonium sulfate precipitation beneficial points : to keep the properties of the antibody molecule, F(ab')2, but performance recorded less antibodies than other purification techniques such as ion exchange chromatography ( ion-exchange chromatography) and affinity chromatography ( affinity chromatography) Ammonium sulfate has very high solubility to 4M concentration, while at a concentration of 3.2M, most of the protein had been precipitated Only 30-60 minutes after dissolving the ammonium sulfate concentration for each selection, the entire corresponding protein was precipitated When the particles resuspended in buffer protein precipitated new, molecular structure and biological activity of the protein remain unchanged Some salts precipitate under the same proteins are removed by dialysis Thus, the protein solution into products (eg antivenom ) has recently been purified condensed, but still retain biological function of antibodies Of course, in addition to the concentration and nature of ammonium sulfate , the precipitated protein depends on the pH and temperature of the solvent At pH = 4.2 and temperature 20°C, with ammonium sulfate concentration = 14% , not all protein antibodies, including Fc and lipids were precipitated, was removed with Whatman filter paper , the filtrate containing F(ab') were collected 31 - The precipitate F(ab ') with ammonium sulfate, filter Whatman filter paper obtained by precipitation, dialysis, and sterile filtration produce antivenom F(ab')2 When the pH of the filtrate adjusted to 6.8/20 oC, then add ammonium sulfate to 36% , mix well, after 60 minutes to precipitate the entire F(ab')2 with ammonium sulfate salt (Table 3:13) Remove ammonium sulfate dialysis rich precipitates from F(ab') Sterile membrane filter with a pore size cellulose acetatae have 0.2 micron filter, create antivenom F(ab') semi Table 3.14 shows that the plasma filter once more to save more Here, a time filter will reduce 25 ml (BC+BM) antivenom F(ab')2, or vials, each vial production costs about 70 USD (Table 3.24), was reduced expenses cost significantly ( by using filters SEIZ, oldfashioned, if the filter with a new machine, in addition to better quality, faster time filtering, even more savings) Table 3:15 shows some of the chemicals used purified antivenom Combined with 3:24 table (Appendix thesis) , that amount of funds for the purification of large no This is a technique easy to implement, simple, small, serving the needs of patients is not large objects Table 3.16 shows the refined 650 ml ( BC+BM) antivenom F(ab') semi -finished products after inspection and satisfactory basis, closing ml vials, 130 vials (BC+BM) multivalent antivenom F(ab')2 product - Determination of antivenom purified polyclonal F(ab')2: ... 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... BC+BM antivenom Bottle 500 ml 650 Polyvalent antivenom F(ab’)2 Vial ml 650 130 3.1.4.3 Determination of purity of Polyvalent antivenom F(ab’)2 Table 3:17: Quantification of protein, albumin,... generated antivenom Table 3:13: Precipitation amount of F (ab'') obtained target Parcel The filtrate is F(ab’)2 precipitation has F (ab '') (ml) obtained (gam) Parcel 2900 210 Parcel 5140 395 Total 8040