抗旋毛虫鸡卵黄抗体的活性检测

目的制备特异性抗旋毛虫的鸡卵黄免疫球蛋白,研究其免疫应答规律及其稳定性。方法用纯化的旋毛虫幼虫免疫育龄种鸡,经水稀释法初步纯化浓缩后获得卵黄抗体。应用酶联免疫吸附试验检测卵黄抗体的效价及其敏感性和稳定性。结果通过免疫可成功诱导出高效价的抗旋毛虫抗体,效价可达1∶106以上,免疫应答持续时间可达34周以上。免疫后卵黄抗体敏感性和血清抗体敏感性无显著性差异(P>0.05)。稳定性试验表明IgY敏感性高,在pH4～10及温度不超过60℃条件下活性稳定。结论成功制备出高效价的特异性抗旋毛虫卵黄抗体,其敏感性高,有一定的耐热、耐酸碱性,可进一步用于旋毛虫感染的研究。     

抗弓形虫特异性鸡卵黄抗体的制备与鉴定

目的　制备高效价特异性鸡卵黄免疫球蛋白 ( yolkimmunoglobulin ,IgY)抗体 ,为弓形虫病防治的研究探索新的方法和途径 ;方法　用提纯的弓形虫RH株抗原免疫育龄种鸡 ,获取大量含高效价IgY的卵黄 ,经水稀释法初步纯化浓缩后 ,用ELISA检定其免疫学活性 ,应用SDS -PAGE分析、鉴定IgY的分子基础 ;结果　获得ELISA效价为 1× 10 7的抗弓形虫IgY抗体 ,经SDS -PAGE分析 ,出现 1条蛋白带 ,其分子量大小根据电泳迁移率计算 ,初步鉴定为 4 6kDa用弓形虫抗原免疫Lyh ern种鸡制备的IgY抗体效价高、特异性强 ;水稀释法初步提纯获得的IgY抗体分子量为 4 6kDa,为在弓形虫病的特异性免疫学诊断、预防和治疗中的应用奠定基础。     

幽门螺杆菌重组VacA-HpaA IgY的制备

目的:采用基因工程技术大量表达、提纯幽门螺杆菌(H pylori)细胞空泡毒素毒性片段,和黏附素片段的融合蛋白,以此为抗原,制备高效价的抗VacA-HpaA蛋黄抗体(IgY)．方法:大量培养重组菌pQE30-VacA-HpaA- DH5α,诱导表达获得融合蛋白VacA—HpaA, Ni2 -NTA树脂纯化．将纯化的重组蛋白免疫鸡,水稀释法提取IgY,硫酸铵沉淀法纯化浓缩VacA—HpaA IgY．ELISA法测定抗体产生的时间-效价变化,SDS—PAGE分析纯度, Bradford法测定蛋白含量,Western blot检测其分别对VacA和HpaA抗原的特异性,ELISA法检测效价．结果:重组蛋白主要以包涵体形式表达,免疫鸡后提取的IgY可与该蛋白发生免疫反应, IgY效价总体上随免疫时间增加而升高．经纯化、浓缩后,获得纯度为60％左右,效价为1: 12 800的VacA-HpaA IgY,蛋白浓度为22 g／L, Westem blot显示在Mr27 000和Mr30 000处分别有相应条带,ELISA检测与VacA和HpaA反应的效价分别为1:3200和1:6400(P<0．01)．结论:成功制备了高浓度、高效价的抗重组VacA-HpaA的特异性IgY．     

日本血吸虫可溶性虫卵抗原经两种免疫途径获得的鸡卵黄抗体IgY动态观察

目的对比两种免疫途径产生抗日本血吸虫可溶性虫卵抗原(SEA)的特异性IgY抗体水平动态变化。方法7只新西兰兔感染日本血吸虫尾蚴(1500/只)42d后解剖收集虫卵,制备SEA。将SEA分别经皮下和静脉注射免疫健康海蓝蛋鸡(3只/组),首次和第2次免疫间隔2周,之后每次间隔4周,共免疫5次,50μg/(只·次)。取两组免疫前,以及首次免疫后2～18周生产的鸡蛋,用水稀释法粗提IgY,ELISA检测每2周IgY的动态变化。IgY纯化试剂盒(EGGstract IgY Purifiction System)纯化静脉组首次免疫后8～18周的IgY,紫外吸收法检测抗体浓度,琼脂糖双扩散法和ELISA检测IgY峰值水平的抗体效价,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析比较免疫前后抗体特异性。结果静脉组和皮下组分别在首次免疫后8和12周IgY抗体水平达高峰,A492值分别为1.28和0.78,静脉组IgY水平至18周仍维持较高水平,皮下组抗体水平达到峰值后逐渐下降。纯化后IgY浓度约为6.5～9.0mg/ml,琼脂糖双扩散法和ELISA测得静脉注射组峰值水平抗体效价分别为1∶16和1∶51200。经SDS-PAGE和Western blotting分析,纯化后的IgY在相对分子质量(Mr)25000和68000处各有一条清晰条带,且免疫后IgY可与SEA发生特异性反应。结论静脉注射法比皮下注射法能获得更高水平的鸡抗日本血吸虫SEA特异性IgY抗体,且纯化后的IgY抗体具有较好的特异性。     

抗旋毛虫肌幼虫ES抗原IgY的制备及鉴定

目的制备抗旋毛虫肌幼虫排泄-分泌(excretory-secretory,ES)抗原的鸡卵黄免疫球蛋白(IgY),测定其效价及用于检测抗原的敏感性。方法 4只24w龄罗曼母鸡用旋毛虫肌幼虫ES抗原经大腿外侧与胸部肌肉免疫4次(首次剂量为500μg/只,加强剂量为250μg/只),每次间隔10d。取免疫前和首次免疫后42d的鸡蛋卵黄,用水稀释法提取IgY,考马斯亮蓝法测定蛋白含量,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot)及间接荧光抗体试验(IFAT)对IgY进行分析,ELISA检测纯化后IgY的效价及检测抗原的敏感性。结果罗曼鸡经ES抗原免疫后,每枚鸡蛋经提纯后均可得到约70mg抗体,SDS-PAGE表明纯化的IgY有2条主要蛋白带,分子量为67kDa、23kDa,Western blot与IFAT发现提纯的IgY可识别肌幼虫虫体与ES抗原。IgY的抗体效价为1∶107,IgY-夹心ELISA检测旋毛虫抗原的敏感性为1.17ng/mL。结论制备的抗旋毛虫ES抗原的IgY具有较高的效价与敏感性。     

抗幽门螺杆菌粘附素HpaA卵黄抗体的制备

目的研制高效价特异性的抗幽门螺杆菌黏附素鸡卵黄抗体免疫球蛋白抗体。方法用纯化重组幽门螺杆菌黏附素(Helicobacter pyloriadhesin A,rHpaA)抗原免疫22周龄罗曼鸡,母鸡免疫应答后,在卵黄中产生抗幽门螺杆菌黏附素抗体;经硫酸铵法初步纯化浓缩后;以间接ELISA鉴定抗体效价及免疫学活性;采用Bradford法测定蛋白含量;并用SDS-PAGE法测定分子量及鉴定抗体纯度。结果母鸡初次免疫第10d即有抗体产生,初次免疫后75d左右抗体效价超过1∶10000;最高可达为1∶12800。卵黄抗体经硫酸铵法纯化后,用SDS-PAGE分析,出现两条蛋白带,纯度达95%以上。其含量为10.56mg/ml蛋黄。结论本研究首次研制并鉴定抗黏附素卵黄抗体,开拓了我国预防幽门螺杆菌感染的新领域,分析了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。有望获得高效价、高产量的抗黏附素的IgY抗体(IgY-HpaA),为制备口服制剂开辟了广阔前景。     

抗大肠杆菌O157鸡卵黄抗体的初步研究

目的以煮沸15min灭活后的大肠杆菌O157为抗原,分4次免疫产蛋鸡,获取高免卵黄。经水稀释法去脂、超滤、盐析等粗分离,DEAESephadexA-50离子交换层析纯化,得到产物IgY为电泳单点纯,且仍保持效价在1∶104以上。从分离纯化过程中的跟踪检测表明,其卵黄制备的IgY抗体产量多、效价高、特异性强,可进一步用于大肠杆菌感染的检测研究。     

抗日本血吸虫SEA鸡卵黄抗体的制备与鉴定

目的 制备特异性抗日本血吸虫可溶性虫卵抗原（SEA）的鸡卵黄免疫球蛋白（IgY）并检测其特异性、敏感性。 方法 用日本血吸虫SEA经翅膀下静脉和皮下免疫25周龄海兰母鸡4次（首次剂量为60 μg/只，加强剂量为30 μg/只），每次间隔10 d。取免疫前和首次免疫后35 d的鸡蛋卵黄，分别用水稀释法提取IgY，BCA法测定蛋白含量，并进行十二烷基磺酸钠?鄄聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹（Western blotting）分析，ELISA检测纯化后IgY特异性和敏感性。分别提取免疫后不同时间的卵黄抗体，观察抗体效价的变化。 结果 海兰母鸡经SEA首次免疫后35 d，每枚蛋经提纯后可得到约61 mg抗体，经SDS-PAGE和Western blotting分析，纯化的IgY有1条主要蛋白带，相对分子质量（Mr）为130 000，并可被SEA识别。母鸡初次免疫后第10天，经SDS-PAGE和ELISA分析，鸡蛋卵黄内即有抗体产生，初次免疫后31 d效价可达1∶1 600。双抗体夹心ELISA结果显示纯化后的IgY有较高的敏感性，可检测到的SEA达2.4 ng/ml。 结论 制备的抗SEA鸡卵黄抗体的特异性和敏感性均较高。     

Experimental manufacture of equine antivenom against yamakagashi (Rhabdophis tigrinus)

Yamakagashi, Rhabdophis tigrinus, is a natricine snake widely distributed in eastern Asia. Severe bite cases, some with fatal outcomes, occur regularly in Japan. Because previous production of R. tigrinus antivenom in rabbits and goats was quite effective, we considered the experimental manufacture of a new antivenom against R. tigrinus in horses. This new antivenom could be used in emergency treatment of snakebite victims. Two horses were immunized with venom extracted from about 500 snakes. After an adequate increase of the antivenom titer, serum was collected and subjected to standard purification procedures for the manufacture of equine antivenoms. The purified immunoglobulin fraction was freeze-dried in 1,369 vials under optimum conditions for therapeutic use. This antivenom proved to be very potent in neutralizing the coagulant and hemorrhagic activities of the snake venom. In cases of severe bites, this antivenom was used and recognized as effective even after the occurrence of severe symptoms.     

Detection of circulating antigen in serum of mice infected with Schistosoma japonicum by immunomagnetic bead ELISA based on IgY

We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis.     

Detection of rabies-specific antigens by egg yolk antibody (IgY) to the recombinant rabies virus proteins produced in Escherichia coli

We obtained rabies-specific egg yolk antibodies (IgY) by immunizing hens with recombinant His-tagged nucleoprotein and phosphoprotein (rN, rP) of the rabies virus (CVS-11 strain) expressed in Escherichia coli. The anti-rN and rP IgY were shown to bind specifically to the respective proteins of the CVS-11 strain of rabies virus by Western blotting, immune fluorescent assay and immunohistochemistry, indicating that IgY to rabies recombinant proteins could serve as a reagent for diagnosis of rabies virus infection.     

卵黄抗体的结构与功能其在人及动物疾病中应用进展

鸡卵黄免疫球蛋白(IgY)又称卵黄抗体,是禽类受特定抗原刺激后分泌的特异性抗体。因具有耐热、耐酸和抗蛋白酶降解等特性,早期开发产品作为食品或食品添加剂应用受到重视,其产品开发在食品安全、生态农业方面有重要社会效益和经济效益,产品由于含针对某病原生物特异性抗体,可抵抗病原生物的感染,因而在动物及人类疾病的防治方面有开发应用价值,在人类健康保健也有十分重要的应用前景。本文就动物和人类抗病毒、抗细菌、抗寄生虫、抗肿瘤等相关的IgY生物制剂产品研发及应用进行综述。     

Present tests for detection of snake venom: clinical applications

Immunologic tests for detection of snake venom and venom antibodies have important clinical applications. Enzyme-linked immunoassay (ELISA) and radioimmunoassay (RIA) provide adequate specificity and sensitivity. The former is much more widely used because it is inexpensive, relatively easy to perform, and uses stable reagents. Some ELISA systems will detect 0.5 ng of venom; however, a sensitivity of 10 to 100 ng is more usual. Minimum running time is 30 to 45 minutes; with longer times, greater sensitivity can be attained. Wound aspirate, serum, and urine are the most suitable materials for venom detection. ELISA has been used for clinical diagnosis of snakebite, to monitor antivenom dose, to study clinical syndromes associated with envenomation, to detect venom in forensic cases, and to evaluate first aid techniques. The indirect ELISA usually is used for detecting and titrating venom antibody. This is potentially useful in epidemiological studies of snakebite incidence, in evaluating potency and paraspecific activity of antivenoms, and in studying response to venom immunogens. Current ELISA systems involving snake venoms have low specificity, and most cannot reliably differentiate venoms of related snakes. Venom antibody detection assays are less satisfactory than those for venom; nonspecific reactions and cross-reactivity are unacceptably high. Methods for improvement of snake venom immunodiagnosis are discussed.     

Oral administration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa infections in patients with cystic fibrosis: a phase I feasibility study

Respiratory infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Chronic Pseudomonas aeruginosa (PA) infections ultimately occur in virtually all patients. It is impossible to eradicate PA when a patient has been chronically colonized. Immunotherapy with specific egg‐yolk antibodies (IgY) may be an alternative to antibiotics for the prevention of PA infections. We wanted to determine if treatment with specific IgY can prolong the period between the first and the second PA colonization? And long‐term, can the treatment diminish the number of positive PA cultures and postpone the onset of chronic colonization? CF patients gargled daily with an IgY‐antibody preparation, purified from eggs of hens immunized with PA bacteria. They were compared to a group of patients who did not gargle with the preparation. Both groups had their first colonization with PA eradicated by antibiotics. The basic treatment was essentially the same in both groups. In the initial study, the period between the first and second colonization with PA was significantly prolonged for the treated vs. the control group (Kaplan‐Meier P = 0.015, Breslow test). In the prolonged study, the treated group had only 2.5 sputum cultures positive for PA per 100 months of observation, and none of these patients became chronically colonized with PA. No adverse events were reported. In the control group, 13.7 cultures per 100 months of observation were positive for PA, and 5 (24%) patients became chronically colonized with PA. This feasibility study shows that antipseudomonal IgY has the potential to effectively prevent PA colonization without any severe adverse effects. A phase III study should be initiated. Pediatr Pulmonol. 2003; 35:433–440. © 2003 Wiley‐Liss, Inc.     

Chicken Egg Yolk Antibodies Specific for the γ Chain of Human Hemoglobin for Diagnosis of Thalassemia

Immunoglobulin Y (IgY) technology was used to generate anti-hemoglobin Bart's (Hb Bart's) IgY antibodies (Abs) for development into an enzyme-linked immunosorbent assay (ELISA) test for thalassemia diagnosis. Hb Bart's purified from the hemolysate of a patient with Hb Bart's hydrops fetalis (homozygous alpha-thalassemia) was used to immunize a chicken via the pectoralis muscle. After water dilution and sodium sulfate precipitation, 40 to 70 mg of IgY could be extracted from an egg. IgY, first detected in sera 2 weeks after immunization, reached the highest titer at week 4, and the titer remained stable for at least 2 weeks before declining. The pattern of Ab response in the yolk was the same as in the serum but was somewhat delayed. The IgY Abs produced reacted with gamma globin, Hb Bart's, Hb F, normal cord hemolysate (Hbs F plus A), and Hb Bart's hydrops fetalis (Hbs Bart's plus Portland) and to a lesser degree with beta globin, Hb A, Hb A2 and adult hemolysate (Hbs A plus A2), but the Abs did not react with alpha globin. Immunoaffinity purification with Hb A coupled to Sepharose was used to isolate an unbound IgY that reacted with Hb F, Hb Bart's, and gamma globin, and this IgY was used to develop an ELISA test for thalassemia diagnosis. The results of direct ELISA analyses of 336 hemolysate samples from individuals with various known thalassemia genotypes and phenotypes and from healthy individuals confirmed the specificity of the polyclonal Abs for Hbs containing Hb F and Hb Bart's. This specificity, which was due to the Abs' strong reactivity in cases of pathologic thalassemic diseases and weak reactivity in cases of nonpathologic thalassemic diseases, depended on the levels of Hb Bart's and Hb F.     

Electrophoretic studies on molecular defects of von Willebrand factor and platelet glycoprotein IIb-IIIa with antibodies produced in egg yolk from laying hens.

Immunoglobulins isolated from egg yolk (IgYs) are a convenient source of polyclonal antibodies. Their purification is simple and the yields important (50 mg immunoglobulins/egg). Nevertheless their biochemical characteristics are different from those of rabbit antibodies generally used for the study of molecular defects of plasmatic and platelet proteins. Provided standardization is achieved, IgYs can be used for immunoelectrophoresis, immunoprecipitation or immunoblotting assays. We describe here the electrophoretic and immunoblotting conditions employed to explore human plasmatic and platelet von Willebrand factor (vWF) and human platelet GP IIb-IIIa using IgYs. These two proteins are involved in primary hemostasis and their absence or abnormality is responsible for hereditary bleeding disorders. The methods were applied to the characterization of patients with vWF or GP IIb-IIIa defects and compared to classical mammalian IgG immunoelectrophoretic techniques. Results were further confirmed by flow cytometric analysis.     

抗日本血吸虫可溶性虫卵抗原IgY的制备及初步应用

目的 目的 制备抗日本血吸虫可溶性虫卵抗原 （Soluble egg antigen, SEA） 鸡卵黄免疫球蛋白 （Egg yolk immunoglobu? lin, IgY）, 探讨其应用于血吸虫病流行区查病的可行性。 方法 方法 在血吸虫病流行区采集间接红细胞凝集试验 （IHA） 阳性 者尿液, 在非流行区采集健康人尿液。以SEA经皮下注射免疫莱杭母鸡获得anti?SEA IgY, 以十二烷基磺酸钠?聚丙烯酰 胺凝胶电泳 （SDS?PAGE） 测定其相对分子量; 并以anti?SEA IgY作为捕捉抗体, 采用双抗体夹心法ELISA检测IHA阳性者 和健康人尿液中的血吸虫循环可溶性虫卵抗原 （Circulating soluble egg antigen, CSEA）。 结果 结果 成功制备并纯化anti? SEA IgY。共检测IHA阳性者尿液48份, 在尿液中检出CSEA阳性26例, 检出率为54.17%; 检测健康人尿液10份, 均为阴 性。 结论 结论 基于IgY的免疫诊断技术可有效检出人尿液中CSEA, 其作为一种便捷、 无创伤的新方法可应用于血吸虫病 查病工作中。     

ELISA confirmation of acute and past envenoming by the monocellate Thai cobra (Naja kaouthia)

The monocellate Thai cobra (Naja kaouthia) is a major cause of snake bite mortality and morbidity throughout Thailand, but neither the local nor the systemic effects of its venom are diagnostic. Species diagnosis is important because only monospecific antivenoms are available for treatment in Thailand. We tested the ability of the ELISA technique to detect venom antigen in the sera of 58 acute snake bite cases including 4 fatalities, and venom antibody in 51 patients bitten between 1 month and 19 years previously. N. kaouthia venom antigen was found in 8 of 33 patients with only local envenoming and in 14 of 20 with local plus systemic (neurotoxic) envenoming, but the mean venom concentration was 33 times greater in the latter group. The serum of 1 fatal case contained banded krait (Bungarus fasciatus) but no cobra venom antigen. N. kaouthia venom antibody was present in sera of patients bitten between 1 month and 7 years previously. Antibody was found in 6 of 8 patients who had had local envenoming alone but in only 19 of 41 who had had systemic envenoming treated by antivenom. The titer of antibody declined with an approximate half time of 2-3 years. One patient had a significant titer of B. fasciatus venom antibody. This study confirms the value of ELISA-immunodiagnosis and the predominance of N. kaouthia as a cause of neurotoxic envenoming in the Bang Phli area. However, the attribution of 1 fatal case to B. fasciatus bite suggests that patients with neurotoxic signs should be given B. fasciatus antivenom if they fail to respond to cobra antivenom.     

Cross neutralization of Hypnale hypnale (hump-nosed pit viper) venom by polyvalent and monovalent Malayan pit viper antivenoms in vitro and in a rodent model

Hypnale hypnale (hump-nosed pit viper) is a medically important venomous snake in Sri Lanka and Southwestern India. Bite of this snake may result in hemostatic dysfunction, acute kidney injury and death. Clinical studies indicated that the locally available polyvalent antivenoms produced in India are not effective against hump-nosed pit viper envenoming. Hence, there is an urgent need to search for effective antivenom. In this paper, we examined the ability of Calloselasma rhodostoma (Malayan pit viper) monovalent antivenom and the Hemato polyvalent antivenom (both produced by Thai Red Cross Society, TRCS) to neutralize the lethality and toxic effects of H. hypnale venom, as C. rhodostoma is considered a sister taxon of H. hypnale. In vitro neutralization studies showed that the Hemato polyvalent antivenom effectively neutralized the lethality of H. hypnale venom (1.52 mg venom/mL antivenom) as well as the hemorrhagic, procoagulant and necrotic activities of the venom. The monovalent C. rhodostoma antivenom could also neutralize the lethality and toxic activities of the venom, but the potency was lower. The Hemato polyvalent antivenom also effectively protected mice from the lethal and local effects of H. hypnale venom in an in vivo rodent model of envenoming. Furthermore, the polyvalent antivenom could also effectively neutralize the venom of Daboia russelii (2.50 mg venom/mL antivenom), another common cause of snake bites in Sri Lanka and South India. These findings suggested that the Hemato polyvalent antivenom may be beneficial in the antivenom treatment of H. hypnale envenoming.     

Dynamics of passive immunity to West Nile virus in domestic chickens (Gallus gallus domesticus)

Birds are the principle amplifying hosts for West Nile virus (WNV), and understanding the acquisition and decay of passive immunity is important to avian surveillance and diagnostics. We characterized passive transfer of WNV-neutralizing antibody from chicken (Gallus gallus domesticus) hens to eggs and chicks and the protective efficacy and decay of maternally acquired antibody over time. We also characterized age-associated changes in magnitude of viremia and examined the possibility of vertical transmission of WNV. All egg yolks and chicks from seropositive hens were maternal antibody positive. Maternal antibodies were undetectable in most chicks by 28 days post-hatch (PH), but some chicks remained protected as late as 42 days PH. By 56 days PH, chicks from immune hens had viremia profiles similar to control chicks. There were significant age-related differences in WNV-attributed morbidity and viremia levels of unprotected chicks. Vertical transmission of WNV was not detected.