| Gloudius intermedius belongs to the Viperidae family, Crotalinae subfamily, Gloydius Genus. It is the most toxic one among six species of the Genus, which is widely distributed in northwestern and northern China. It is closely related to local people's lives. Snake venom, as a mixed secretion in the venom glands, contains many components, when act on different target molecules/cells, may bring about several toxication effects, including muscle damage, bleeding, edema, blood pressure decline, etc.In 1979, researches from Zhao-er Mi found that domestic anti-pit-viper serum didn't neutralize venom of G. intermedius, indicating that there was obvious difference between the venom composition of G. intermedius and five other species in this Genus(G. brevicaudus, G. strauchii, G. saxitilis, G. ussuriensis and G. shedaoensis).Due to the broad distribution in northern China and the severe fatalness, up to now little is known about the tissue damage in envenomation, and the lack of specific anti-venom serum impedes the effective treatment of snakebites.Materials and Methodsⅰ)The tissue damages induced by the snake venom, including the paw edema of mice, muscle damage and creatine kinase release after subcutaneous injection of crude venom, blood pressure change after ear intravenous injection of the crude venom in rabbit.ⅱ)The preparation of anti-venom serum and the protection of toxication. After emulsifying crude venom with Freund's adjuvant as antigens to immunize New Zealand white rabbits (initial immunization, five times enhancing immunization), collected rabbit anti-serum, the anti-serum titer and effect value were assessed by double immune diffusion and indirect ELISA test, the binding specificity were analyzed by Western blot.ⅲ)Toxication protection assay. Mixing and incubating lethal dose of crude venom with different ration of anti-serum in vitro, intraperitoneally injected the mixture into mice and evaluated the protection efficacy according to survival rate in 24 hours.Results are as following:ⅰ) 1.5 h after injection, the paw edema arised. It even became notable in 3 hours.ⅱ) Compared with control,12 hours after intraperitoneally infection of the venom into mice, myofibril bundles were hydrolyzed and destroyed. Muscle fibers arrangement showed serious disorder. As to the myocardial injury, some filaments were dissolved, the space between myofibrils increased.ⅲ) 3h-6h after intravenously tail injection of mice, the CK activity of the serum gradually increased, the maximum activity increased to 1.174U/mL(7.2 times compared with the that from beginning), later CK activity decreased gradually.ⅳ) 90min after intraperitoneally injection of the crude venom into rabbit, the blood pressure decreased rapidly, the blood pressure even dropped to the pressure of 40% of that of the beginning in 120min. Compared with saline control, the difference was significant (P< 0.01).ⅴ) Double immunodiffusion tests in agar showed that antibodies diluted to 2 times,4 times and 8 times caused clearly visible precipitation line, at 16 times the precipitation line has blurred, indicating that at the fifteenth week after immunization, high titer antibodies produced in the rabbit blood.ⅵ) Indirect ELISA assay showed that the anti-serum titers was above 1:128000. Western blotting revealed that positive reaction corresponded to toxins in three protein ranges, molecular weight 14.4kD-18.4kD, around 41kD and 70kD, which means that the principal proteins elicited the immune system to produce the antibodies.ⅶ) After neutralized in vitro, above 0.2mL rabbit anti-serum exhibited markedly protective effect against challenge of 0.76mg/kg venom in mice. more than half mice survived.It means that the prepared antiserum possessed detoxifying activity and significantly reduced the mortality.Conclusion:Our work has established good basis for the exploitation of effective anti-serum for the treatment of the snakebites in the future. |
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