双价抗蛇毒鸡卵黄抗体制备与生物活性研究

目的 通过双价抗蛇毒鸡卵黄抗体(bivalent anti-snake venom immunoglobulin yolk,双价IgY)的制备及其相关特性研究,为多价抗蛇毒IgY的制备和应用奠定基础.方法 两种单一抗原(舟山眼镜蛇、圆斑蝰泰国亚种)按顺序依次交替注入单只鸡体内,水稀释法制备双价IgY;测定双价IgY效价(间接ELISA法)、交叉免疫特性(双向免疫扩散试验)及对溶膜活性(溶膜试验)、半数致死量(LD50)的中和作用.结果 初次免疫后第28-42天,以水稀释法提取的双价IgY对眼镜蛇毒及蝰蛇毒的效价分别为1:12 800和1:6400.双价IgY与眼镜蛇亚科、蝰业科6种蛇毒有明显的交叉免疫反应,而与蝮亚科4种蛇毒的交叉免疫反应不明显.双价IgY能显著降低眼镜蛇、蝰蛇毒对鸡卵黄膜的溶膜作用,也能显著延长眼镜蛇和蝰蛇伤小鼠的存活时间(P<0.05),同等剂量的双价lgY提高眼镜蛇伤存活率优于蝰蛇伤.结论 双价IgY能够显著中和眼镜蛇、蝰蛇毒的溶膜和致死活性,能有效保护机体免受眼镜蛇毒和蝰蛇毒的攻击.     

抗眼镜蛇毒鸡卵黄抗体的制备、纯化和保护性实验研究

目的:探索抗眼镜蛇毒鸡卵黄抗体(IgY) 的制备方法及其生物学活性，为替代马源性抗蛇毒血清奠定基础。 方法:眼镜蛇毒原毒免疫母鸡,采用水稀释-硫酸铵盐析-阴离子交换层析三级提取法纯化IgY；SDS-PAGE 测定各级提取物的抗体纯度；间接ELISA 法和双向免疫扩散法观察效价变化；动物体外中和实验检测其生物活性。 结果:卵黄经水稀释法及经60% 硫酸铵盐析蛋白回收率为40.36%；进一步经阴离子交换层析，总蛋白回收率14.86%，效价提高24倍；IgY分子量约为220 kD，由两个亚基组成；体外中和试验表明，2.28 mg/kg 特异性IgY能完全中和2 LD50 的眼镜蛇毒，保护小鼠免受眼镜蛇毒的攻击。 结论:以眼镜蛇毒原毒为免疫源，经水提、盐析、层析三级提取，可从鸡卵黄中获得高纯度、高活性的特异性IgY。     

新型抗蝰蛇毒血清对不同蝰蛇毒作用的实验研究

目的探讨新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的作用. 方法采用免疫电泳、SDS-聚丙烯酰胺电泳及动物实验等方法. 结果国产圆斑蝰蛇毒的LD50(腹腔注射)为0.306±0.003mg/kg,缅甸蝰蛇毒的LD50(腹腔注射)为0.290±0.003mg/kg.免疫电泳结果表明国产圆斑蝰蛇毒和缅甸蝰蛇毒均和新型抗蝰蛇毒血清产生明显的免疫沉淀线.体外中和实验表明抗蝰蛇毒血清对国产圆斑蝰蛇毒的中和抗体效价为2750μg/ml(约450LD50), 对缅甸蝰蛇毒的中和抗体效价为2490μg/ml(约430LD50).体内保护实验表明给予0.05ml抗蝰蛇毒血清可抵御254μg(约41.5LD50)国产圆斑蝰蛇毒或116μg(约20LD50)缅甸蝰蛇毒的攻击,使小鼠全部存活. 结论新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的中和抗体效价均较高,有较大的应用价值.     

蝰蛇毒压电免疫传感器固定方法的研究

目的：为研制蝰蛇毒压电免疫传感器，研究抗蛇毒抗体固定于石英晶体银电极表面的固定技术。方法：采用马抗蝰蛇毒血清抗体和抗蝰蛇毒鸡卵黄抗体作为生物敏感材料，对比研究了胱胺自组装-PSS反相吸附法和PEI粘附-戊二醛交联法：比较了采用两种固定方法所制的压电免疫传感器的性能。结果：鸡卵黄抗体采用PEI粘附-戊二醛交联法效果较好，其制备的IgY压电免疫传感器检测蝰蛇毒灵敏度为0．5ug／mL；而马血清抗体用胱胺自组装-PSS反相吸附法较好，其制备的IgG’免疫传感器检测蝰蛇毒灵敏度为10ug／mL。结论：以PEI粘附-戊二醛交联法固定抗蝰蛇毒鸡卵黄抗体所制备的蝰蛇毒压电免疫传感器的性能稳定，特异性好，可实现蛇毒的快速检测。     

抗眼镜王蛇毒鸡卵黄抗体的研制及初步应用

目的　研制抗眼镜王蛇毒鸡卵黄抗体 ,并研究该抗体在鸡卵黄中表达过程。方法　以甲醛减毒处理的眼镜王蛇粗毒免疫莱航母鸡 ,母鸡免疫应答后 ,在卵黄中产生抗眼镜王蛇毒抗体 ,应用嗜硫色谱柱HiTrapIgYPurificationHP从鸡卵黄中提取抗眼镜王蛇毒抗体 ;以间接ELISA法及双向免疫扩散测定抗体的效价、特异性及免疫活性 ;采用Lowry氏法测定蛋白含量 ;采用SDS PAGE法测定相对分子质量 (Mr)及鉴定抗体纯度。结果　母鸡初次免疫第 9天即有抗体产生 ,初次免疫 6 0d后抗体效价超过 1∶10 0 0 0 0 ,卵黄抗体经嗜硫色谱柱纯化后 ,经SDS PAGE检测为电泳纯 ,纯化后抗体效价较纯化前提高 4倍 ,每枚蛋中平均可获得较纯抗体 97.5mg。动物体外中和试验表明 ,特异性IgY能有效中和眼镜王蛇毒 ,阻止眼镜王蛇毒对小白鼠的攻击。结论　研制并鉴定了抗眼镜王蛇毒鸡卵黄抗体 ,分析抗体在产蛋期卵黄液中表达的进度 ,为今后该抗体的持续、高质量诱导奠定基础 ,同时也促进其他抗蛇毒鸡卵黄抗体的研究开发及蛇毒单克隆抗体的研制     

抗白色念珠菌IgY的稳定性及体内抗感染活性研究

目的观察抗白色念珠菌IgY的稳定性及体内抗感染活性。方法水稀释法从免疫鸡卵黄提取抗白色念珠菌IgY,酶联免疫吸附试验间接法和微量间接血凝试验测定其抗体效价,并观察其对热、酸碱度、胰蛋白酶及冻存时间的稳定性和对小鼠腹腔感染白色念珠菌的保护效果。结果水稀释法提取的抗白色念珠菌IgY效价为1∶5120;IgY在25~70℃处理15min,pH3.0~11.0、37℃处理2h,或普通冰箱冻存6年效价改变不明显;在37℃下与胰蛋白酶作用2h效价仍保持50%。IgY对免疫功能低下小鼠腹腔感染白色念珠菌有明显的保护作用(P<0.01)。结论抗白色念珠菌IgY具有良好的稳定性和较好的体内抗感染活性。     

卵黄抗体用于眼镜王蛇毒抗原检测的初步研究

眼镜王蛇属于眼镜蛇科、王蛇属，是世界上个体最大的毒蛇，排毒量最大，致死、致残高，早确诊早治疗是降低该类毒蛇咬伤、致死、致残的关键。在蛇伤诊断及蛇毒检测方面，国外主要采用ELISA，由于其使用的诊断试剂中的抗体来源于马等哺乳动物，而这些哺乳动物源性IgG类抗体易产生假阴性或假阳性结果，降低诊断的准确性。卵黄抗体，又称IgY，不仅在预防、治疗人和动物的某些疾病中具有广阔前景，而且由于IgY不会与病人血液样品中类风湿因子、补体系统等发生反应，因而使得其在免疫诊断、生化检测等领域的应用上更具有开发的潜力。国外文献报道制备了针对响尾蛇毒、竹叶青蛇毒等多种蝰蛇科蛇毒的卵黄抗体。     

新型抗蝰蛇毒血清对不同喹蛇毒作用的实验研究

目的　探讨新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的作用 .方法　采用免疫电泳、SDS -聚丙烯酰胺电泳及动物实验等方法 .结果　国产圆斑蝰蛇毒的LD50 (腹腔注射 )为 0 .3 0 6± 0 .0 0 3mg/kg ,缅甸蝰蛇毒的LD50 (腹腔注射 )为 0 .2 90± 0 .0 0 3mg/kg .免疫电泳结果表明国产圆斑蝰蛇毒和缅甸蝰蛇毒均和新型抗蝰蛇毒血清产生明显的免疫沉淀线 .体外中和实验表明抗蝰蛇毒血清对国产圆斑蝰蛇毒的中和抗体效价为 2 75 0 μg/ml(约45 0LD50 ) ,对缅甸蝰蛇毒的中和抗体效价为 2 490 μg/ml(约 43 0LD50 ) .体内保护实验表明给予 0 .0 5ml抗蝰蛇毒血清可抵御 2 5 4μg(约41 5LD50 )国产圆斑蝰蛇毒或 116μg(约 2 0LD50 )缅甸蝰蛇毒的攻击 ,使小鼠全部存活 .结论　新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的中和抗体效价均较高 ,有较大的应用价值 .     

鸡卵黄抗体的制备以及金磁微粒为载体去除人血浆部分高丰度蛋白的研究

目的:以人血浆白蛋白(HSA)和IgG为免疫原,制备特异性鸡卵黄抗体IgY(Egg yolk immunoglobulin),并将其固定于金磁微粒表面,用于HSA和IgG的去除研究.方法:用HSA、IgG以及混合成分分别作免疫原免疫Roman母鸡.制备抗HSA和IgG鸡卵黄抗体IgY,并对IgY的分离提取条件进行优化.SDS-PAGE和间接ELISA检测IgY的纯度和效价.将高效价IgY固定于金磁颗粒表面进行血浆高丰度蛋白去除研究.结果:免疫后60～120 d内,鸡血清抗体效价可达1∶15 000～1∶25 000;收获鸡蛋,提取得到的卵黄抗体IgY效价可达1∶10000～1∶25000,纯度98%以上;采用金磁微粒载体固定IgY,可对血浆中的HSA,IgG进行特异性的去除.结论:人血浆中的高丰度蛋白成分HSA和IgG免疫产蛋母鸡后,可从鸡卵黄中分离提取到高效价、高纯度的卵黄抗体IgY;IgY偶联于金磁微粒表面可特异性的去除人血浆中的HSA和IgG,作为血浆蛋白质组学研究的一种新方法,有较好的应用前景.     

IgY治疗烧伤后肠源性白色念珠菌感染的实验研究

目的　为治疗严重烧伤后肠源性白色念珠菌感染寻求新的治疗措施。方法　选用标准白色念珠菌菌株免疫纯系Leghorn鸡 ;采用改良水溶法 (WD)从蛋黄中提取抗体IgY ,双紫外光测定抗体含量 ,通过SDS PAGE电泳检测抗体纯度 ,Wes tern blotting免疫印迹法测定该抗体来源 ,ELISA法检测热处理后IgY的活性 ,采用纯系SPF小鼠烧伤 (2 5 %Ⅲ度烧伤面积 )肠源性白色念珠菌感染的模型 ,特异IgY治疗并与对照组比较。结果　IgY的效价高 (1∶12 80 0 )、特异性强和理化性质稳定 ;喂服IgY治疗烧伤小鼠后 ,明显抑制小鼠肠道内白色念珠菌的黏附、生长 ;明显降低了烧伤小鼠血浆中二胺氧化酶 (DAO)和肿瘤坏死因子 (TNF α)的释放 ,维护肠黏膜上皮的完整 ,减轻了白色念珠菌对小肠上皮细胞的损害 ;促进肠淋巴细胞分泌IgA ;恢复肠道局部免疫功能。结论　特异IgY效价高、特异性强、理化性质稳定 ,治疗烧伤小鼠肠源性白色念珠菌感染疗效显著     

Clinical profile,species-specific severity grading,and outcome determinants of snake envenomation: An Indian tertiary care hospital-based prospective study

Objective:

We undertook this study to assess the clinical profile and outcome determinants of different snake envenomation as well as to assign species-specific severity grade to different cases based on clinico – laboratory evidence scale.

Materials and Methods:

A prospective clinico – epidemiologic evaluation for outcome determinants of snakebite envenomation was carried out based on a clinico – laboratory severity grading scale, among 76 patients over a period of 2 years, in a tertiary care hospital in southern India.

Results:

Majority of patients were male agricultural workers (53.9%) followed by housewives (19.7%), and students (9.2%). Occurrence of viper snake envenomation with hemotoxic syndrome (73.68%) was highest followed by cobra and krait envenomation with neurotoxic (19.73%) and hemo – neurotoxic (5.3%) syndrome, respectively. On the contrary, maximum mortality and severity was seen in krait (60%) followed by cobra (13.33%) and viper (8.9%) envenomation. The average dose of anti-snake venom (ASV) administered varied from 9.83 (±7.22) to 20.25 (±4.92) vials throughout grade I to IV in all snake species envenomation. An increase in severity grade, ASV dose, and mortality were observed with the corresponding delay in ‘bite to needle time.’ Also, initial traditional treatments and krait species envenomation were significantly associated with higher grades of severity and mortality.

Conclusion:

There is an urgent need to spread awareness among the community for avoidance of traditional treatment and any delay in medical intervention in snakebite incidents.     

Clinical and biological surveillance of envenomed patients

Faced with an envenomation, the problem is to take sufficiently rapidly the decision to administer the only effective treatment--immunotherapy--, to know which antivenom to choose and how long to administrate it. If the snake is not identified, symptoms and initial development give information on the type of venom. It is convenient to classify the symptoms according to four clinical types: i) the cobra syndrome with a potentially fatal evolution within two to ten hours and which resembles an Elapid bite, ii) the viper syndrome associating bleeding and inflammation, which can be due either to a viper, pit viper or, in Australia, to Elapids, iii) disturbance of blood circulating functions and iv) disturbance of other live functions. Between the third to the half of snakebite victims present no envenomation. Severe envenomations must be monitored in an intensive care unit, with experience in emergency management and monitoring of patients with major life-threatening conditions. Throughout the world, snakebites induce more than 100,000 deaths every year. Schematically, the emergency may be considered in terms of seconds for blood circulation disorders, minutes for respiratory paralysis, and hours for the coagulopathy.     

Amino acid changes following intraperitoneal administration of Tityus zulianus scorpion venom in mice. Study with subcutaneous microdialysis and capillary electrophoresis

Scorpion human envenoming is a public health hazard in the southwest of Venezuela. Tityus zulianus is one of the scorpion species whose venom causes lung edema and cardiac failure in children. These occasionally deadly manifestations have been attributed to a massive sympathetic discharge. The intraperitoneal administration of T. zulianus venom (20 micrograms/g mouse) to anesthetized mice during subcutaneous microdialysis caused increased secretions, dyspnea, seizures and death between 30 min to 2 h. Seven amino acids were analyzed by capillary electrophoresis with laser induced fluorescence detection (CE-LIFD) in the collected samples before and after the venom administration. We found an increase of arginine (39%), phenylalanine (40%) and glutamate (94%), with no changes in valine, serine and aspartate, changes were significant when the injection of venom and vehicle were compared and before vs after venom injection. Further investigation is needed to know if the observed changes could be related to the molecular mechanisms of the venom or some of its components and therefore with the envenoming symptoms. To our knowledge, this is the first report with subcutaneous microdialysis and CE-LIFD coupling in scorpion envenomation studies in vivo, in mice.     

Role of nitric oxide in the local and systemic pathophysiological effects induced by Bothrops asper snake venom in mice

Objective To assess the role of nitric oxide in the most relevant local and systemic manifestations in mice injected with the venom of the snake Bothrops asper. Mice were pretreated with nitric oxide synthase inhibitors, and the modifications of the pathological effects induced by the venom were tested. Results Inhibition of NO synthesis did not affect acute local myonecrosis and hemorrhage in muscle tissue upon intramuscular injection of venom. Local footpad edema was reduced in mice pretreated with the NO synthase inhibitor L-NAME, and a reduction in the extent of inflammatory infiltrate in muscle tissue was observed after envenomation in mice pretreated with L-NAME and aminoguanidine. The most pronounced effect of NOS inhibition by L-NAME was an increment in the lethal activity of the venom, when injected by the intraperitoneal route. Conclusion Nitric oxide does not seem to play a significant role in the local acute pathological alterations (hemorrhage and myonecrosis) induced by B. asper venom in mice, although it contributes to edema and inflammatory infiltrate. Nitric oxide exerts a protective role in the systemic pathophysiological manifestations leading to lethality. Received 7 November 2005; returned for revision 19 December 2005; returned for final revision 3 February 2006; accepted by M. Katori 11 February 2006     

Nephrotoxic action of rattlesnake and sea snake venoms: an electron-microscopic study.

The fine structure of renal corpuscles and proximal convoluted tubules of the right kidneys of Suiss mice were studied 9 hr after the injection of the venom of Crotalus atrox (Western diamond-back rattlesnake) or the venom of Laticauda semifasciata (broad-banded blue sea snake). Rattlesnake envenomation resulted in several ultrastructural changes in the renal corpuscles and proximal convoluted tubules. Visceral epithelial changes included intracellular oedema, blebbing, vesiculations, the formation of microvillus projections and dilatation of the endoplasmic reticulum and mitochondria. Changes in the parietal epithelium were similar except that no microvillus projections were noted. Mesangiolysis was a consistent finding. Collagenous fibrils were very prominent in the lysed areas of the mesangial cells. Increased numbers of lysosome-related structures were noted in the proximal convoluted tubule cells. Most of the nuclear cisternae of the cells of the renal corpuscles and proximal convoluted tubules were greatly dilated. Sea snake envenomation resulted in focal organellar swelling and focal intracellular oedema of the visceral epithelium. This venom did not affect the cells of the proximal convoluted tubule. The presence of light and dark visceral epithelial cells has been a consistent finding for both the control and the environmated groups.     

皖南地区眼镜蛇毒活性组分镇痛效应的初步研究

 目的：从皖南地区眼镜蛇毒粗毒中分离纯化出活性组分眼镜蛇毒镇痛因子（cobra venom analgesic factor, CVAF),并研究其镇痛效应。方法：采用阳离子交换色谱（cation-exchange chromatography, CEC）和分子排阻层析（size-exclusion chromatography, SEC）从眼镜蛇粗毒中分离纯化出镇痛活性组分CVAF,SDS-聚丙烯酰胺凝胶电泳法和毛细管区带电泳法进行纯度及相对分子量的鉴定;将小鼠随机分为生理盐水正常对照组和盐酸吗啡阳性对照组、CVAF实验组,采用小鼠热板法和醋酸扭体法评估活性组分CVAF的镇痛效应。结果：纯化后得到活性产物为单一组分,相对分子量为6 500 D;热板法显示吗啡组在给药0.5 h后达高峰,6 h镇痛作用消失;活性组分CVAF的镇痛作用自用药后0.5 h开始,且持续8 h仍存在。扭体法中0.03 mg/kg、0.1 mg/kg和0.3 mg/kg的活性组分在给药后小鼠扭体抑制率分别为27%、50%和70%;0.3 mg/kg实验组与吗啡组相比无明显差异（P> 0.05）。结论：从皖南地区眼镜蛇粗毒中分离纯化出的活性组分CVAF具有剂量依赖性镇痛效应。     

Hypersensitivity to airborne spitting cobra snake venom.

BACKGROUND: Although the cytolytic, neurotoxic, and hemolytic actions of snake venoms are well known, the ability of airborne inhaled snake venom of the spitting cobra to induce asthma in snake handlers has not been reported. OBJECTIVE: To report the allergenicity of inhaled snake venom in a snake handler who developed increasing hypersensitivity to airborne venom, produced by spitting cobras during public demonstrations. METHODS: Serum samples were obtained from 2 handlers (our study patient and another snake handler who reported developing wheezing when handling spitting cobras), and desiccated venom was obtained from 9 species to which the handlers were exposed. Serum from an asymptomatic and nonatopic snake handler exposed to the same snake species was used as a control. Phosphate-buffered saline extracts were prepared from the desiccated venom, proteins in the venom extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting was performed. Inhibition enzyme-linked immunosorbent assays (ELISAs) were performed to demonstrate cross-reactivity. RESULTS: The study patient had never been previously bitten by a cobra. Wheezing occurred rapidly on inhalational exposure and was reversed by inhalation of salbutamol. The patient had developed IgE antibodies to 9 different snake venoms on Western immunoblots, with major IgE binding proteins of 59 to 63 kDa and 8 to 15 kDa. The cross-reactive nature of the IgE epitopes in the venoms in the different species was also confirmed by 50% inhibition of IgE binding in an ELISA by preincubation with unrelated species. Life-threatening sensitivity of the patient was sustained after a long period of avoidance. CONCLUSIONS: We propose that aerosolized snake venom be considered a new potential source of allergens that may result in anaphylaxis on subsequent exposure. Further studies of the development of specific IgE sensitization following snakebites and the risks of such sensitization should be conducted on snake handlers, particularly those who demonstrate the spitting species.     

Antibody from mice immunized with DNA encoding the carboxyl-disintegrin and cysteine-rich domain (JD9) of the haemorrhagic metalloprotease, Jararhagin, inhibits the main lethal component of viper venom

Envenoming by the Brazilian pit viper, Bothrops jararaca, induces extensive local and systemic haemorrhage in humans. The severe and occasionally lethal outcome of envenoming is prevented only by administration of antivenom which is conventionally prepared by hyperimmunization of large animals with an individual venom or a range of venoms. Since snake venoms typically consist of numerous molecules, only some of which are toxic, antivenoms are antigenically crude preparations whose therapeutic value would theoretically be enhanced by restricting antibody specificity to toxic venom molecules. We report here that high-titre IgG antibody from mice immunized by the GeneGun with DNA encoding the carboxy-terminal JD9 domain of Jararhagin, a haemorrhage-inducing metalloprotease in B. jararaca venom, extensively neutralized the main lethal component of B. jararaca venom. This is to our knowledge the first study to apply DNA-based methods to preparation of antivenom; it represents a novel approach with greater immunological specificity and fewer hazards than conventional systems of antivenom production.