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. 2. Assessing the efficacy for detection venoms and venomous snake species identification of ELISA kit in clinical specimens. The main new scientific contributions of the thesis: This research has succeed in development of an AB-ELISA kit for detection venoms of four snake species: Trimeresurus albolabris, Naja naja kaouthia, Calloselasma rhodostoma and Ophiophagus hannah. This is the first time in Vietnam, antivenoms produced by IVAC in Nha Trang city (products clinically used for treatment of snake bite) have been used to develope the AbE-ELISA for detection venoms of two species: Trimeresurus albolabris and Naja naja kaouthia. Both AB-ELISA and AbE-ELISA have ability for detection of standard snake venom samples at levels lower than 4 ng/ml, depending on sample types such as blood, plasma, urine, buffer and type of snake venoms. AbE-ELISA kit has ability for detection of Trimeresurus Albolabris and Naja kaouthia in all kind of assay samples in clinical, include: whole blood, urine and blister fluid. The layout of the thesis: The thesis consists of 112 pages with 19 figures, 3 diagrams, 10 charts and 15 tables, in which: retionale (2 pages); Introduction (33 pages); Introduction (33 pages); Materials and menthods (22 pages); Result (27 pages); Discussion (25 pages); Conclusion (2 pages); Recommendation (1 page); References 122 documents (Vietnamese 12, English 110). 3 CHAPTER 1: LITERATURE REVIEW 1.1. ACCIDENT AND DIAGNOSIS OF VENOMOUS SNAKE BITE 1.1.1. Common venomous snakes causing snake bite accident in 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. 1.3.2. Designs of ELISA applied in developing tests for detection of snake venom - General principle of ELISA test: the use of antigen or antibody as marker to detect antigen-antibody complexs by color indicator. - Design of sandwich ELISA using two antibodies pattern Design of sandwich ELISA avidin-biotin (AB-ELISA): consist of capture antibody, detection antibody conjugated biotin, HRP conjugated avidin and substrate solution to detect antigen-antibody complexes. - Design of sandwich ELISA using three antibodies pattern Design of sandwich AbE-ELISA using three antibodies pattern: consist of one capture antibodies, detection antibodies is an antigen specific antibodies but have different productive origins with capture antibodies. HRP conjugated antibodies that specific to detection antibodies and substrate solution to detect antigen – antibody complexs. In this design of test, detection antibodies have not conjugated enzyme or biotin so the stability and efficacy of this are better than detection antibodies used in the above design. HRP conjugated antibodies that specific to detection antibodies can be supplied by commercial company so it has higher specific and stability. 5 1.3.3. Criteria in evaluation of an ELISA In development of ELISA for detection of snake venom, evaluation of criterias are often interested in specificity, sensitivity, stability and testing time. 1.3.4. The commercial ELISA kit for detection of snake venom Up to now, there are two snake venom detection kits that was developed in Australia and India. However, these are not suitable for detection of venomous snake venom in Vietnam. 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 processing of immunized like the protocol of Chotwiwatthanakun and et al (2001) is a low dose, low volume, multi-site injection along the spine tip and booster. 2.2.2. Indirect ELISA assay - Identify specific antibodies titer of anti-snakevenom serum - Assessment of cross reactivity between mono-specific anti- snakevenom serum and venoms of other snakes 2.2.3. Purification of antibodies Antibodies specific for each species of snake venoms was extracted and purified by three steps purification process: * Step 1: extraction IgG from rabbit serum by affinity chromatography with protein G column. * Step 2: extraction venom specific IgG from total IgG by immune – chromatography with column contains specific snakevenom antigen. * Step 3: remove the antibodies molecules that cross-reactivity between snake species by immune adsorbtion menthod with allergic snakevenom antigen column. 2.2.4. Biotinylation of antibodies 7 2.2.5. Preparation of mono-specific horse antiserum against Trimeresurus albolabris and Naja kaouthia supplied by IVAC 2.2.6. AB-ELISA kit for detection of snakevenom 2.2.6.1. Design AB-ELISA kit The kit is built on the principle of sandwich ELISA with Avidin- Biotin complex . 2.2.6.3. Protocols of AB-ELISA assay - Step 1: AB-ELISA kits are mounted on racks. - Step 2: add 100 µl sample (whole blood, plasma, serum, urine, blister ). - Step 3: add 100 µl biotin conjugated detection antibodies. - 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 2.2.7.1. Design AbE-ELISA kit The kit is built on the principle of sandwich ELISA with 3 specific antibodies (horse antibody, rabbit IgG antibody and HRP conjugated mouse antibody that anti rabbit IgG). 2.2.7.3. Protocols of AbE-ELISA assay Consists of 5 steps similar to those in the section 2.2.6.3 Test validation: - Positive: OD sample > X OD negative control + 3SD - Negative: OD sample ≤ X OD negative control + 3SD 2.2.7.6. Evaluating the stability and efficiency of AbE-ELISA kit in the laboratory in detecting snakevenom Similar to the method described in section 2.2.6.4 2.2.8. Assessing the efficacy for detection venoms and venomous snake species identification of ELISA kit in clinial specimens * Step 1: Complete the document of clinical trial research and collected sample from the patients were envenomed by snakebite. * Step 2: Evaluate effectiveness of detection for venomous snake venom by done the AbE-ELISA assay in the process described in section 2.2.7.3 for all collected samples. * Step 3: The evaluation of suitability between snake bite species confirmed diagnosis by ELISA kit and snake bite species confirmed diagnosis by clinical base on KAPPA coefficient. 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. [...]... 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. .. stability of AB -ELISA kit A major limitation that we encountered when making AB -ELISA kit for detection of venomous snake venom is stability time of this kit less than three months 4.2 AbE -ELISA KIT FOR DETECTION VENOM OF 2 SNAKE SPECIES Trimeresurus albolabris and Naja kaouthia 4.2.1 AbE -ELISA kit for detection venom of 2 snake species Trimeresurus albolabris and Naja kaouthia The AbE -ELISA kits are... model of AbE -ELISA kit for detection of snake venom in this study is consistent with the model of 3 antibodies sandwich 19 ELISA assay of Selvanayagam (1999) and Hung Dong Zong (2003) 4.2.3 The sensitivity of AbE -ELISA assay The sensitivity of AbE -ELISA assay also at ng/ml level and the lowest in buffer solution is 0,5 – 1 ng/ml depend on the type of snake venoms The sensitivity of AbE -ELISA assay for... IDENTIFICATION OF ELISA KIT IN CLINICAL SPECIMENS 4.3.2 The result of AbE -ELISA assay for detection venoms and venomous snake species identification in clinial specimens - The result of AbE -ELISA assay in specimens: blood, urine and blister In this study, AbE -ELISA assay was detected venomous snake venom of all types of clinical specimens as whole blood, urine and blister - The result of AbE -ELISA assay... samples is 2/4 = 50% 15 Bảng 3.12 The results AbE -ELISA assay for detection of venom of T albolabris along with clinical diagnosis to identify species of snake bite Clinical Bitten by No bitten by ELISA assay T albolabris T albolabris Sum Positive 45 (62,5 %) 1 (2 %) 46 Negative 27 (37,5 %) 49 (98 %) 76 72 (100%) 50 (100 %) 122 Sum KAPPA = 0,56 Comment: The positive results of AbE -ELISA assay for detection... snake venom of 3 antibodies sandwich ELISA kit manufactured by Selvanayagam in 1999 and Hung DongZong 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... 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... Cross-reactivity of AbE -ELISA assay with venoms of other venomous snakes in Vietnam Chart 3.8 Cross-reactivity of AbE -ELISA assay with venoms of other venomous snakes at concentration 25 ng/mL Comment: at all concentrations examined, OD values of wells that specific reaction are significantly higher than the remaining wells as negative control wells and cross-reactivity wells 3.2.5 The stability of AbE -ELISA kit... 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... 18 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 . IVAC 2.2.6. AB -ELISA kit for detection of snakevenom 2.2.6.1. Design AB -ELISA kit The kit is built on the principle of sandwich ELISA with Avidin- Biotin complex . 2.2.6.3. Protocols of AB -ELISA assay -. 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. an ELISA In development of ELISA for detection of snake venom, evaluation of criterias are often interested in specificity, sensitivity, stability and testing time. 1.3.4. The commercial ELISA