Study On Detection Of Stonefish Toxins And Preparation Of Antitoxins

A wide variety of marine organisms can secret protein toxins, including cnidarians(such as sea anemones and jellyfish), molluscs(such as conus and aplysia), echinoderms(such as starfish and sea urchins), venomous fishes(Acanthotoxic fishes) as well as sea snakes. Stonefish(Synanceia) is one of the most toxic fishes in the oceans, and is mainly found in the tropical waters of the Pacific and the Indian Ocean. Synanceia verrucosa is one of the most toxic venomous fishes and is widely distributed in the South China Sea. The dorsal, anal and pelvic fins of stonefishes have venomous spines, and venom sacs containing lethal and hemolytic protein toxins are allocated on both sides of the dorsal fins. After being stung by a stonefish, the wounds get severe symptoms, such as severe pain, low blood pressure, edema, nausea, dizziness, chest tightness, respiratory and circulatory failure or even death. Hundreds of cases of stonefish injuries are reported in foreign countries each year, dozens of cases have been reported in China, and serious symptoms such as cellulitis and tissue necrosis occurred.Antivenom treatment is needed for severe marine protein toxin poisoning. At present, antivenoms against sea snakes, sea anemones, jellyfish and stonefish have been reported. The only stonefish antivenom was developed by Australian Commonwealth serum laboratories, which have been used in the treatment of stonefish toxin poisoning. 8 mice monoclonal antibodies induced by Stonustoxin(SNTX) which was extracted from Synanceja horrida, have high affinity and neutralization activity. Due to the lack of stonefish antivenoms, symptomatic and supportive therapy is the major treatment of stonefish toxin poisoning in China, such as pain relief, anti infection, anti shock and traditional Chinese medicine treatment, etc. Therefore, it is very important to develop an effective antivenom for stonefish poisoning treatment. The establishment of the detection method is also necessary to determine whether it is stonefish poisoning.There are now some researches on detection methods of protein toxins. The main techniques include bioassay method, mass spectrometry, protein chip technology, gene analysis method, immunoassay method and colloidal gold strip rapid detection method. Bioassay methods are to determine the nature of biological toxins by using animal’s sensitivity to toxins, mouse bioassay method and rat bioassay method are commonly used. Protein chip technology belongs to the technical field of high-throughput protein analysis, which can be used to investigate the protein interaction, the protein-nucleic acid interaction and drug targets. Mass spectrometryies are used to identify proteins by comparing a mass spectrometry peak diagram and a two stage mass spectrometry peak diagram of digested protein fragments with the theoretical ones, this method has been widely used in detections of marine polypeptides and protein toxins.Immunoassay method is a high sensitivity detection method based on the catalytic activities of enzymes to substrates and the specific reactions of antigen-antibodies. The method includes radioimmunoassay, immunofluorescence and enzyme linked immunosorbent assay(ELISA) and has the advantages of high selectivity, low detection limit and simple operation. ELISA method not only can be used for the detections of antigens, but also can be used for the detections of antibodies. The colloidal gold detection technology is a rapid detection technology, in which colloidal gold shows color and binds proteins without changing protein biological activities. In addition, the detection with colloidal gold test strips takes only a few minutes or more, and is simply operated. Therefore, it has been widely used in medical science, food safety and other fields.Recently, stonefish toxin neo VTX has been found from the crude venom in our laboratory, the c DNA sequences of neo VTX α and β subunit were cloned, and the α subunit was also successfully expressed in prokaryotic cells. Based on the previous work, this research is to perform the stonefish toxin purification, antibody preparation and the establishment of ELISA method and colloidal gold test strips for detection of stonefish toxins in human plasma, and to lay the foundation for the diagnosis and treatment of stonefish poisoning. The main results of this paper are as follows:1. Preparations of stonefish toxin neo VTX and horse antibody with high activity. The AKTA prime protein purification system was used to separate neo VTX from stonefish crude venom, and 322.6 mg of neo VTX(the purity was 88.53%) was successfully prepared. In order to provide the basic experimental data for the preparation of neo VTX mouse monoclonal antibody and horse antibody, the immuning conditions of Balb/C mice using the crude venom and the purified neo VTX were investigated. SDS-PAGE or size-oclusion HPLC showed that the purity of horse antitoxins was high(87.05%). The antibody titer of mouse monoclonal antibody(dissolved in PBS to 1 mg/ml) was 6400, and the horse antibody titer reached 409600(40 mg/ml). The neutralizing activities of the mouse monoclonal antibodies against neo VTX was poor, 30 times of 3 LD50 neo VTX(w/w) displayed no protective effect. However, the horse antibody had high neutralizing activity. All mice survived when neo VTX(3 LD50) and horse antibody(1:3(w/w))( i.v.) or stonefish crude venom(4 LD50) and horse antibody(1:2(w/w)) were injected i.v.). In addition, 30 min afte the adminstrtion of neo VTX(32 LD50)(i.p.), 3 times of neo VTX horse antibody(1:3, w/w))(i.v.) could protect all mice.2. The ELISA method could sensitively detect neo VTX in plasma. Horse antibody and HRP were coupled with sodium periodate method. The optimized experimental conditions were explored through a series of experiments, including the coating concentration of horse antibody and the optimum concentration of horseantibody-HRP. A double antibody sandwich ELISA detection method for neo VTX in PBS solution and in human plasma was established and a standard curve was made. neo VTX can be quantitively analyzed in the range of 4-512 ng/ml in PBS with the linear regression equation of Y=1.676X-1.140(r2= 0.9728, n= 8, p< 0.0001). When neo VTX samples were detected in human plasma, the OD value was significantly lower than the same concentration of neo VTX in PBS solution. Therefore, the standard curve in human plasma was established, and neo VTX in plasma was well detected in the range of 4-2048 ng/ml with linear regression equation of Y=0.3094X-0.1208(r2= 0.9907, n = 10, p< 0.0001). The method is suitable for the analysis of neo VTX in PBS and human plasma with high sensitivity and specificity.3. The colloidal gold detection method of neo VTX was established. The colloidal gold solutions of 6 different sizes were parpared with sodium citrate method using chloroauric acid as a raw material. The ultraviolet-visible spectrophotometer and electron microscopy demonstrated the colloidal gold had apprirate size. The best p H value(8.0) and marked quantity of horse antibody(96 μg/ml) were obtained, the suitable materials of colloidal gold chromatography were chosen as follows:NC Membrane selection was type Millipore 135, colloidal gold pad was type GL 0194, the sample pad was type GL-b01, water filter was type H5076. Then, the suitable concentration of samples were selected, the goat anti-horse Ig G was diluted 45 times and spotted on the control line, the horse antibody was spotted on the test line at a concentration of 4 mg/ml. the gold-labeled horse antibody was diluted 2 times and coated on colloidal gold pads. Finally, the colloidal gold test strips were prepared successfully and showed high sensitivity, specificity and stability. The test strip could achieve a sensitivity of 100 ng/ml for neo VTX and showed no response to BSA, ovalbumin and jellyfish toxin.