| Background:According to resource, the antibody of anti-snake venom can be divided into two types --- antivenene and anti-snake venom immunoglobulin yolk. The former was isolated from mammalian blood and the latter was extracted from avian egg yolk. From 1920s, foreign scholars had prepared several kinds of anti-snake venom IgY, using cobra, Russell's viper, Saw-scaled viper, B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambica venoms to immunize chickens and neutralization of these anti-snake venom IgY to relevant snake venoms had obvious effects. In domestic, there had been reported about the preparation and characteristic research of anti-snake venom IgY of Ophiophagus Hannah, Agkistrodon Halys, cobra, and viper venoms. Compared with other antibodies extracted from mammalian blood, such as rat, rabbit, procine, horse, bovine, et al, IgY has extreme superiority: IgY was not combining with rheumatoid factor and protein A or C, not activating complements system; The characters of IgY above could reduce non-specific reaction, avoiding the influence of glutaraldehyde to immunological detection; Moreover, it was convenient to product eggs with low cost, which were suitable for large-scale production; The egg yolk of hen immunized contained only one kind of antibody (IgY) with good homogeneity and physico-chemical stability; And IgY also contained properties of acid resistance, heat resistance, easily preserved, convenience for transportation and application; Then, because of distant phylogenetic relationship of amphibian and chicken, immune response was easily generated with little stimulation. It was needed to prepare polyvalent anti-snake venom IgY against more kinds of snakes with highly toxic in south china, for prevention and treatment. It was not necessary to classify snakes to cure snakebite, seizing the time saving, reducing the mortality rate and possibility of serious pathological change. Based on the preparation of univalent anti-snake venom IgY against cobra and viper in this laboratory before, the research of bivalent anti-snake IgY will supply a new approach for snakebite.Objective:Preparing bivalent anti-cobra and viper venom immunoglobulin from egg yolk (IgY), detecting their bioactivity and observing the protection effect of anti-cobra and viper venom IgY by oral administration against cobra and viper envenomation in mice, provided a new approach for prevention and treatment of snakebite, establishing foundation for exploring substitute anti-venom from horse.Methods:一The preparation of bivalent anti-snake venom IgY1 The preparation of bivalent anti-snake venom IgY With optimized immunization programs, cobra and viper venom antigens were prepared by the patent snake venom utensil. Freund's adjuvant and a small dose of cobra venom (or viper venom) antigens were mixed with 1:1 (V/V) by emulsifier made of our own, into water-in-oil emulsion particles. At the first immunization, Freund's complete adjuvant was used to emulsify snake venom, and then Freund's incomplete adjuvant was used. Booster immunization of each antigen was respectively taken a time at the third and fifth week after the first immunization, and immune dosage was increased gradually and intermittently. Two kinds of single antigen alternately injected into leghorn hen (at multiple subcutaneous sites of wings, breast, abdomen, back and muscles). Injection interval was one week.2 Specific IgY from yolk was identified by double immunodiffusion and immunoelectrophoresis. The purity IgY products were measured by SDS-PAGE. The concentration of specific IgY and its dynamic variety in plasma were observed by indirect ELISA assay.3 The purification of bivalent anti-snake venom IgY Egg yolk was separated the from egg albumen by Yolk Segregation and Collection Utensil, which was invented of our own. IgY was extracted by water dilution assay. 二The characterization of bivalent anti-snake venom IgY1 The cross immune response and its intensity with 10 common snake venom in China were detected by double immunodiffusion method.2 Both cobra and viper venom had yolk membrane lysis effect. The neutralization of snake venom and anti-snake venom IgY was detected by yolk membrane lysis detector, invented of our own.3 The protection effect of anti-snake venom IgY by intraperitoneal injection against snake venom envenomation in mice was detected after they were neutralized in vitro.三Animal protection experiment of bivalent anti-snake venom IgY1 The potency of bivalent anti-snake venom IgY in plasma were observed by indirect ELISA assay at different time by intragastric administration.2 The protection effect of bivalent anti-snake venom IgY by intragastric administration was detected by intraperitoneal injecting snake venom in mice at different time.四Statistic T-test and SPSS software were adopted to statistic and analysis experimental data.Results:1 The snake venom prepared by Snake Venom Utensil with high activity and less foreign matters. After the first immunization with little dosage of cobra and (or) viper venom, specific IgY was detected 9 days later. The potency of anti-snake venom IgY increased continuously with intermittent immunization and gradually increasing dosage, and maintaining high potency for a longer time.2 The Yolk Segregation and Collection Utensil, made of our own, was adopted to separate egg yolk. The yolk in this way was smooth, glossy, integrity and with less residue, benefiting for the next purification.3 The total protein recovery rate of IgY fraction by water dilution method(IgY-WD)reached about 40%. Then, IgY could be higher purified with thiophilic adsorption chromatography, hydrophobic chromatography and gel chromatography.4 By double immunodiffusion assay, immune response of bivalent IgY and Naja atra Cantor, Ophiophagus Hannah venom were the most significant with the clearest precipitation lines. The immune response of bivalent IgY and several snake venoms of Elapinae and Viperinae had cross reactions in different degree. The immune response of bivalent IgY and Crotalinae venom, such as deinagkistrodon, agkistrodon and several others, had no obvious cross reactions.5 Based on the data of yolk membrane lysis detector, some dosage anti-snake venom IgY from IgY-WD had protection effect for egg yolk against cobra and viper venoms.6 Based on the data of neutralization reaction in vitro, some dosage anti-snake venom IgY from IgY-WD completely protected mice against cobra and viper venoms.7 Anti-snake venom IgY in different dosage had different protective effect against cobra and viper venoms in mice.Conclusions:1 The hens was immunized to prepare specific IgY of high potency with low dosage cobra and viper venom in the initial immunization and increasing dosage intermittently and gradually.2 The Yolk Segregation and Collection Utensil and the yolk membrane lysis detector, both of which invented of our own, isolated egg yolk industrially or in batches and actually detected the lysis of snake venoms or other toxins.3 By IgY-WD precipitation assay, IgY with high activity was withdrawn from egg yolk. The IgY with high activity and recovery rate was obtained by IgY-WD assay, which were easy and economic.4 The protective effect and application scope of anti-snake venom IgY in oral administration could be widen by increasing antigens and purified IgY with higher potency. |
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