抗钩虫特异性卵黄抗体的制备与鉴定及其抗钩蚴作用研究
|
摘要
目的钩虫病是全球发展中国家常见的肠道寄生虫病,反复药物驱虫导致耐药危险性增加,而药物的毒副作用也限制了化疗药物在孕妇及婴儿患者中的使用。因此,单纯依靠驱虫药物的防治方法已经不能完全适应现阶段全球钩虫病的防治工作,安全、有效的抗钩虫病替代药物或者功能性食品有待于研制。本课题收集钩虫病患者驱虫后排出的钩虫成虫,制备钩虫成虫抗原(AWA),免疫25w产蛋海兰母鸡,制备抗钩虫成虫特异性卵黄抗体(IgY)并鉴定。动态监测抗体效价,大量纯化效价较高阶段IgY,通过体内、体外试验分析特异性IgY的抗蚴作用,为进一步研制抗钩虫药物或功能性食品奠定基础。
方法应用饱和盐水漂浮法加水洗沉淀法筛查安徽医科大学第一附属医院住院患者23人,收集4位粪检阳性患者药物驱虫后1-5d全程粪便,水洗过筛收集成虫,制备AWA保存备用。购买未经免疫的海兰母鸡雏鸡,养至25W产蛋。每只母鸡以200ugAWA进行皮下加肌肉多点注射免疫,初次免疫后28d给予第二次免疫,间隔10d进行第三次免疫。收集免疫前、后鸡蛋,标记后4℃保存备用。水稀释法粗提抗钩虫IgY抗体,盐析法纯化抗体,SDS-PAGE和Western-blotting进行鉴定。间接ELISA法测定抗体效价,选取效价最高时间段的鸡蛋,大量提取纯化IgY抗体4℃保存备用。采用改良加藤厚涂片法(Kato-Katz法)检获安徽省蒙城县重度钩虫感染患者,收集患者粪便,分别进行固体培养基滤纸法钩蚴培养和试管滤纸法钩蚴培养。分别收集钩虫三期幼虫(L3),进行体外L3期钩蚴与30%,50%和70%浓度的IgY抗体分别共培养以及小鼠腹腔内L3期钩蚴与IgY抗体共培养。倒置显微镜下观察体外共培养后钩蚴形态,并进行钩蚴存活率统计分析。透射电镜观察小鼠腹腔内抗体共培养钩蚴形态,倒置显微镜下根据中性粒细胞黏附程度的不同对钩蚴进行分级计数,统计学分析。利用IgY抗体和FITC标记的抗鸡IgY二抗进行间接免疫荧光实验,观察钩虫成虫抗原在钩蚴体表的分布特点。
结果制备的钩虫AWA浓度为1.21mg/ml。水稀释粗提、盐析法纯化得到的IgY,经Lowry法测得的浓度为1.8mg/ml。SDS-PAGE检测IgY分子量与理论值一致。Western-blotting显示,免疫后的IgY与AWA可以相互识别,而免疫前的IgY则不能识别成虫抗原蛋白。间接ELISA法测定抗体效价,初次免疫后10d左右产生抗体,随着时间推移,抗体滴度逐渐上升,至免疫后55d达到最高峰,效价达到1:10000以上。不同产蛋母鸡之间抗体滴度存在个体差异。用EGG stractTM IgY Purification System纯化试剂盒大量纯化IgY,经Lowry法测得的浓度在5.9-10.1mg/ml之间。钩蚴与IgY体外共培养,钩蚴体表形成絮状或颗粒状附着物,絮状物缠绕虫体,使虫体活动受限;钩蚴存活率统计学分析表明:50%及70%浓度的IgY抗体能够对钩蚴的存活产生影响。小鼠腹腔内钩蚴与抗体共培养显示,IgY抗体能够增加中性粒细胞对钩蚴的趋化、黏附作用。透射电镜观察发现,虫体表膜肿胀、粗糙,横纹间相互的挤压,表膜及肌层电子密度不均匀。免疫荧光实验显示,钩蚴表膜上存在着与钩虫成虫相同的抗原组分,利用成虫粗抗原免疫母鸡得到的IgY抗体可与钩蚴表膜上的抗原结合。
结论本研究成功大量制备了抗钩虫成虫特异性IgY抗体,具有良好的安全性和稳定性,易于保存。我们发现IgY抗体可与钩蚴表膜抗原结合,能够增加中性粒细胞对钩蚴的趋化、黏附作用;IgY抗体对虫体表膜有损伤作用,高浓度IgY抗体影响钩蚴的存活率。表明,特异性IgY抗体对钩蚴具有损伤作用,可用于钩虫病的被动免疫。
Objective: Ancylostomiasis is the common intestinal parasitic diseases in developing countries all over the world. Since repeated deworming leads to a potential risk of drug resistance as well as the drug toxicity and side-effect limits the application of deworming drugs in pregnant woman and infant, the treament depending on anthelmintic can not satisfy the demands on preventing and curing global ancylostomiasis..The safe and effective substitute drug or functional food needs to be developed. In this study, we prepared the adult worm antigen(AWA) using the ancylostomes obtained from the patients’feces who had taken anthelmintic to expel the parasite. Then the ancylostome specific yolk immunoglobulin (IgY) was prepared and identified from the 25-week old HY-line hens immunized by AWA. We monitored the dynamic a ntibody titer of IgY,and purified the antibody at higher titer. The IgY was culture with the third-stage infective larvae(L3) in vitro and in vivo to investigate the effect of IgY upon ancylostome and to lay the foundation for the production of anti-hookworm drugs or functional foods.
Methods: Twenty-three patients from the first affiliated hospital of AnHui medical university were screened by brine flotation and sedimentation method. The dejecta from 4 patients with positive kopr-test was collected for consecutive five days after treated with anthelmintic, and the ancylostomes were sieved to produce AWA. The 25 week-old egg-laying HY-line hens were immunized with 200μg AWA by subcutaneous injection and intramuscular injection.On the day 28th and the day 38th after initial immunization, the hens were immunized repeatedly.The eggs layed before and after immunization were collected to make anti-AWA IgY antibody(Storage Temperature is 4℃).IgY was extracted from the eggs by WD (water-dilution) method and purified by dialysis and salting out. The IgY was analyzed by SDS-PAGE and Western-blotting. Indirect ELISA was used to determine the titer of the antiserum. Large amount IgY was purified while the antibody had a high titer. The patients with severe ancylostomiasis infection in Mengcheng County, Anhui Province diagnosed by Kato-Katz were included in the following experiment. Their dejecta were cultured by solid medium-filter paper cultivation of ancylostoma caninum larvae and tube-filter paper cultivation and the L3 were isolated. The L3 was cultured with IgY at the concentration of 30%, 50% and 70% respectively in vitro or in the abdominal cavity of mice.The larvae cultured in vitro were observed by inverted microscope and their survival rate was calculated. The larvae obtained from the mice were observed by transmission electron microscopy(TEM) and graded according to the neutrophil adhesion by inverted microscope. The distribution of ancylostome antigen on L3 was observated by indirect immunofluorescence staining using anti-IgY and FITC conjugated chicken IgY antibody.
Result: The concentration of AWA was 1.21mg/ml.The concentration of IgY obtained by Lowry method was 1.8mg/ml. The molecular size of IgY detected by SDS-PAGE was equal to its predictive value. Western blotting results showed that the AWA could be recognized by IgY collected from immunizsed hens while not by IgY collected before immunization. Using indirect ELISA, the facts were observed that the IgY antibody began to produce by hens on the tenth day after the initial immunization, and the antibody titer increased gradually over time while reached the peak with a titer overtoped 1:10000 on the 55th day after immunization. There is a discrepancy on IgY antibody titer between different hens. The IgY was purified heavy by EGG stractTMIgY Purification System and the concentration ranged from 5.9 mg/ml to 10.1mg/ml. A floc or granular attachment formed on the surface of L3 after culture in vitro. The activity of L3 had been restricted by attachments. Statistical analysis showed that the IgY with the concentration of 50% or 70% could decrease the livability of L3. It was found that the IgY antibodies could increase chemotaxis and adhesion of neutrophil through the in vivo experiment.TEM observation showed that L3 exhibited a damage of its surface including swelling, roughness, uneven electron density, and the transverse striations of L3 squeezed each other.A same antigen component of adult worm existed on the superficial membrane of L3 disclosed by indirect immunofluorescence.The purified IgY could react with antigen on the cuticular surface of L3.
Conclusion: We have successfully prepared the IgY antibody specific to ancylostome with good security and stability. The yield of IgY is generous and easy to store.The IgY can react with antigen on cuticular surface of L3 and increase chemotaxis and adhesion effect of neutrophil. The IgY bined to L3 may damage its superficial membrane and the antibody with high concentration can decrease the livability of L3.In short, the specific IgY is harmful to larvae, and can be used for immunotherapy against ancylostomiasis.
方法应用饱和盐水漂浮法加水洗沉淀法筛查安徽医科大学第一附属医院住院患者23人,收集4位粪检阳性患者药物驱虫后1-5d全程粪便,水洗过筛收集成虫,制备AWA保存备用。购买未经免疫的海兰母鸡雏鸡,养至25W产蛋。每只母鸡以200ugAWA进行皮下加肌肉多点注射免疫,初次免疫后28d给予第二次免疫,间隔10d进行第三次免疫。收集免疫前、后鸡蛋,标记后4℃保存备用。水稀释法粗提抗钩虫IgY抗体,盐析法纯化抗体,SDS-PAGE和Western-blotting进行鉴定。间接ELISA法测定抗体效价,选取效价最高时间段的鸡蛋,大量提取纯化IgY抗体4℃保存备用。采用改良加藤厚涂片法(Kato-Katz法)检获安徽省蒙城县重度钩虫感染患者,收集患者粪便,分别进行固体培养基滤纸法钩蚴培养和试管滤纸法钩蚴培养。分别收集钩虫三期幼虫(L3),进行体外L3期钩蚴与30%,50%和70%浓度的IgY抗体分别共培养以及小鼠腹腔内L3期钩蚴与IgY抗体共培养。倒置显微镜下观察体外共培养后钩蚴形态,并进行钩蚴存活率统计分析。透射电镜观察小鼠腹腔内抗体共培养钩蚴形态,倒置显微镜下根据中性粒细胞黏附程度的不同对钩蚴进行分级计数,统计学分析。利用IgY抗体和FITC标记的抗鸡IgY二抗进行间接免疫荧光实验,观察钩虫成虫抗原在钩蚴体表的分布特点。
结果制备的钩虫AWA浓度为1.21mg/ml。水稀释粗提、盐析法纯化得到的IgY,经Lowry法测得的浓度为1.8mg/ml。SDS-PAGE检测IgY分子量与理论值一致。Western-blotting显示,免疫后的IgY与AWA可以相互识别,而免疫前的IgY则不能识别成虫抗原蛋白。间接ELISA法测定抗体效价,初次免疫后10d左右产生抗体,随着时间推移,抗体滴度逐渐上升,至免疫后55d达到最高峰,效价达到1:10000以上。不同产蛋母鸡之间抗体滴度存在个体差异。用EGG stractTM IgY Purification System纯化试剂盒大量纯化IgY,经Lowry法测得的浓度在5.9-10.1mg/ml之间。钩蚴与IgY体外共培养,钩蚴体表形成絮状或颗粒状附着物,絮状物缠绕虫体,使虫体活动受限;钩蚴存活率统计学分析表明:50%及70%浓度的IgY抗体能够对钩蚴的存活产生影响。小鼠腹腔内钩蚴与抗体共培养显示,IgY抗体能够增加中性粒细胞对钩蚴的趋化、黏附作用。透射电镜观察发现,虫体表膜肿胀、粗糙,横纹间相互的挤压,表膜及肌层电子密度不均匀。免疫荧光实验显示,钩蚴表膜上存在着与钩虫成虫相同的抗原组分,利用成虫粗抗原免疫母鸡得到的IgY抗体可与钩蚴表膜上的抗原结合。
结论本研究成功大量制备了抗钩虫成虫特异性IgY抗体,具有良好的安全性和稳定性,易于保存。我们发现IgY抗体可与钩蚴表膜抗原结合,能够增加中性粒细胞对钩蚴的趋化、黏附作用;IgY抗体对虫体表膜有损伤作用,高浓度IgY抗体影响钩蚴的存活率。表明,特异性IgY抗体对钩蚴具有损伤作用,可用于钩虫病的被动免疫。
Objective: Ancylostomiasis is the common intestinal parasitic diseases in developing countries all over the world. Since repeated deworming leads to a potential risk of drug resistance as well as the drug toxicity and side-effect limits the application of deworming drugs in pregnant woman and infant, the treament depending on anthelmintic can not satisfy the demands on preventing and curing global ancylostomiasis..The safe and effective substitute drug or functional food needs to be developed. In this study, we prepared the adult worm antigen(AWA) using the ancylostomes obtained from the patients’feces who had taken anthelmintic to expel the parasite. Then the ancylostome specific yolk immunoglobulin (IgY) was prepared and identified from the 25-week old HY-line hens immunized by AWA. We monitored the dynamic a ntibody titer of IgY,and purified the antibody at higher titer. The IgY was culture with the third-stage infective larvae(L3) in vitro and in vivo to investigate the effect of IgY upon ancylostome and to lay the foundation for the production of anti-hookworm drugs or functional foods.
Methods: Twenty-three patients from the first affiliated hospital of AnHui medical university were screened by brine flotation and sedimentation method. The dejecta from 4 patients with positive kopr-test was collected for consecutive five days after treated with anthelmintic, and the ancylostomes were sieved to produce AWA. The 25 week-old egg-laying HY-line hens were immunized with 200μg AWA by subcutaneous injection and intramuscular injection.On the day 28th and the day 38th after initial immunization, the hens were immunized repeatedly.The eggs layed before and after immunization were collected to make anti-AWA IgY antibody(Storage Temperature is 4℃).IgY was extracted from the eggs by WD (water-dilution) method and purified by dialysis and salting out. The IgY was analyzed by SDS-PAGE and Western-blotting. Indirect ELISA was used to determine the titer of the antiserum. Large amount IgY was purified while the antibody had a high titer. The patients with severe ancylostomiasis infection in Mengcheng County, Anhui Province diagnosed by Kato-Katz were included in the following experiment. Their dejecta were cultured by solid medium-filter paper cultivation of ancylostoma caninum larvae and tube-filter paper cultivation and the L3 were isolated. The L3 was cultured with IgY at the concentration of 30%, 50% and 70% respectively in vitro or in the abdominal cavity of mice.The larvae cultured in vitro were observed by inverted microscope and their survival rate was calculated. The larvae obtained from the mice were observed by transmission electron microscopy(TEM) and graded according to the neutrophil adhesion by inverted microscope. The distribution of ancylostome antigen on L3 was observated by indirect immunofluorescence staining using anti-IgY and FITC conjugated chicken IgY antibody.
Result: The concentration of AWA was 1.21mg/ml.The concentration of IgY obtained by Lowry method was 1.8mg/ml. The molecular size of IgY detected by SDS-PAGE was equal to its predictive value. Western blotting results showed that the AWA could be recognized by IgY collected from immunizsed hens while not by IgY collected before immunization. Using indirect ELISA, the facts were observed that the IgY antibody began to produce by hens on the tenth day after the initial immunization, and the antibody titer increased gradually over time while reached the peak with a titer overtoped 1:10000 on the 55th day after immunization. There is a discrepancy on IgY antibody titer between different hens. The IgY was purified heavy by EGG stractTMIgY Purification System and the concentration ranged from 5.9 mg/ml to 10.1mg/ml. A floc or granular attachment formed on the surface of L3 after culture in vitro. The activity of L3 had been restricted by attachments. Statistical analysis showed that the IgY with the concentration of 50% or 70% could decrease the livability of L3. It was found that the IgY antibodies could increase chemotaxis and adhesion of neutrophil through the in vivo experiment.TEM observation showed that L3 exhibited a damage of its surface including swelling, roughness, uneven electron density, and the transverse striations of L3 squeezed each other.A same antigen component of adult worm existed on the superficial membrane of L3 disclosed by indirect immunofluorescence.The purified IgY could react with antigen on the cuticular surface of L3.
Conclusion: We have successfully prepared the IgY antibody specific to ancylostome with good security and stability. The yield of IgY is generous and easy to store.The IgY can react with antigen on cuticular surface of L3 and increase chemotaxis and adhesion effect of neutrophil. The IgY bined to L3 may damage its superficial membrane and the antibody with high concentration can decrease the livability of L3.In short, the specific IgY is harmful to larvae, and can be used for immunotherapy against ancylostomiasis.
引文
1.全国人体重要寄生虫病现状调查办公室.全国人体重要寄生虫病现状调查报告[J].中国寄生虫学与寄生虫病杂志,2005;23(5):332-340.
2.詹斌,肖树华,李铁华,等.钩虫流行现状及疫苗研制进展[J].中国寄生虫学与寄生虫病杂志,2000 ;18(3):182-185.
3.Brooker S,Bethony J,Hotez PJ.Hotez. Human Hookworm Infection in the 21st Century.J Adv Parasitol,2004;58:197-288.
4.胡锡敏,林绍雄,王善青,等.海南省人体钩虫感染情况现状调查分析[J].寄生虫与医学昆虫学报,2007;14(3):145-148.
5.谢红,田洪春,唐中玖,等.四川省钩虫等肠道线虫病2005-2006年监测数据分析[J].寄生虫病与感染性疾病,2008;6(1):21-24.
6.Hotez PJ. Hookworm disease in children.Pediatr Infect Dis ,1989;8(8):516-520.
7.杨毅.钩虫病所致上消化道大出血2例报告[J].江西医药,1999;33(4):236-237.
8.徐明琪.婴儿钩虫病62例临床分析[J].四川医学,2001;22(1):35.
9.罗秀亭,张平安,钱铃.钩虫病误诊32例[J].临床军医杂志,2006;34(3):371-372 .
10.杨松,钩虫病误诊支气管哮喘、慢性阻塞性肺疾病1例分析,医药世界,2009;11(7):343-344.
11.潘卫庆.寄生虫生物学研究与应用[M].北京:化学工业出版社,2007:266.
12.Albonico M,Smith PG,Ercole E,et a1.Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area. Trans R Soc Trop Med Hyg,1995;89(5):538-41.
13.Pritchard DI,Quinnell RJ,Slater AF,et al.Epidemiology and immunology of Necator americanus infection in a community in Papua New Guinea:humoral responses to excretory-secretory and cuticular collagen antigens.Parasitology,1990;100(2):317-326.
14.SorghoH,Bahgat M, Poda J N, et al. Serodiagnosis of Schistosoma mansoni infections in an endemic area of Burkina Faso: performance of several immunological tests with different parasite antigens. Acta Trop,2005;93(2):169-180.
15.Albonico M,Smith PG,Ercole E,et al. Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area.Trans R Soc Trop Med Hyg,1995;89(5):538-541.
16.Prichard R.Genetic variability following selection of Haemonchus contortus with anthelmintics .Trends Parasitol,2001;17(9):445-53.
17.Fincham JE, Markus MB, Adams VJ.Could control of soil-transmitted helminthic infection influence the HIV/AIDS pandemic.Acta Tropica,2003;86(2-3):315-33.
18.杨平生.咪唑类驱虫药致脱髓鞘脑病21例临床分析[J].医学临床研究,2004;21(7):748-750.
19.Alan D.T.Barrett,Lawrence Stanberry.Hookworm Infection. Vaccines for Biodefense and Emerging and Neglected Diseases[M],new york:Academic Press,2009:1365-78.
20.詹斌,肖树华,李铁华,等.钩虫流行现状及疫苗研制进展[J],中国寄生虫学与寄生虫病杂志,2000 ;18(3):182-185.
21.Suzuki N,Lee YC,Site-specific N-glycosylation of chicken serum IgG.Glyeobiology,2004;14(3):275-292.
22.FangYM,KenaW,Curran et a1.Polyclonal Gene Specific IgY Antibodies for Proteomies and Abundant Plasma Protein Depletion[M].New York:Frontiers of Biotechnology & Pharmaceuticals ,2003;4(5):134-141.
23.Schade R,Hlinak A.Egg yolk antibodies,state of the art and future prospects. ALTEX,1996;13(5):5-9.
24.Hatta H,Tsuda K,Ozeki M,et al.Passive immunlzation against dental plaque formation in humans:effect of a mouth rinse containing egg yolk antibodies (IgY)specific to streptccoccus mutarm.Caries Res,1997;31(4):268-274.
25.Sarker SA,Casswall TH,Juneja LR et al. Randomized,Placebo-controlled,clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children withrotavirus diarrhea.Pediatr Gastroenterol Nutr,2001;32(1):19-25.
26.聂荣庆,吴东风,胡国柱,等.特异性IgY抗体对中性粒细胞吞噬功能的影响[J].中国兽医杂志,2004;40(12):23-26.
27.Humphrey BD,Calvert CC,Klasing KC.Klasing The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos),Developmental and Comparative Immunology,2004;28(7-8):665–672.
28.Lee EN,Sunwoo HH,Menninen K,et al.In vitro studies of chicken egg yolk antibody (IgY) against Salmonella enteritidis and Salmonella typhimurium. Poultry Science,2002;81(5):632~641.
29.Akita EM,Nakai S.Comparison of four purification methods for the production of immunoglobulins from egs laid by hens immunized with an enterotoxigenic E.coli strain[J].J Immunol Methods,1993;163(1):141-142.
30.许正敏,李智山,孙莉,等.犬钩蚴固体培养基滤纸培养法[J].中国寄生虫学与寄生虫病杂志,2007;25(4):358.
31.Xiao Shuhua,Peter J,Hotez,etal.Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae .Parasitology International,2001;50(4):241-248.
32.SorghoH,Bahgat M, Poda J N, et al. Serodiagnosis of Schistosoma mansoni infections in an endemic area of Burkina Faso: performance of several immunological tests with different parasite antigens. Acta Trop,2005;93(2):169-180.
33.朱新产,王金宝,王宝维.鸡鸭鹅免疫球蛋白的研究进展[J].中国预防兽医学报,2003;25(6):503-508.
34.Bizhanov G,Vyshniauskis G.A comparison of three methods for extracting IgY from the egg yolk of hens immunized with Sendai virus.Vet Res Commun.2000;24(2) :103-13.
35.Mine Y,Kovacs-Nolan J.Chicken egg yolk antibodies as the rapeutics in enteric infectious disease:a review. J Med Food,2002,5 (3):159-169.
36.Davalos-Pantoja L,Ortega-Vinuesa JL,Bastos-Gonzalez D,etal.A comparative study between the adsorption of IgY and IgG on latex particles.J Biomater Sci Polym Ed ,2000 ,11(6) :657-673.
37.Shin J H ,Yang M ,Nam SW ,et al. Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection.Clin Diagn Lab Immu-nol ,2002 ,9 (5) :1061-1066.
38.Erhard MH,Ozpinar H,Bilal T,et al.The humoral immune response and the productivity of laying hens kept on the ground or in cages.Altern Lab Anim,2000;28(5):699-705.
39.Schade R,Staak C,Hendriksen C,et al.The production of avian(Egg yolk)antibodies:IgY.The report and recommendations of ECVAM workshop21.ATLA,1996;24(6):925-934.
40.常山,张雅萍,查晓霞,等.抗白色念珠菌鸡卵黄抗体(IgY)的研制[J].第三军医大学学报,2002;24(9):1026-1028.
41.Lee KA,Chang SK,Lee YJ et al .Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution.Biochemist ry and Molecular Biology,2002;35 (5):488-493.
42.Kricka LJ.Human anti-animal antibody interferences in immunological assays.Clin Chem,1999;45(7):942-956.
43.Lee EN,Sunwoo HH,Menninen K,et al.In vitro studies of chicken egg yolk antibody (IgY) against Salmonella enteritidis and Salmonella typhimurium.Poultry Science,2002;81(5):632-41.
44.Humphrey BD,Calvert CC,Klasing KC.The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos).Dev Comp Immunol,2004;28(7-8):665-72.
45.聂荣庆,吴东风,胡国柱,等.特异性IgY抗体对中性粒细胞吞噬功能的影响[J],中国兽医杂志,2004;40(12):23-25.
46.郭冠英,杨路.IgY牙膏对远缘链球菌体外黏附及超微结构的影响[J].中国药物与临床,2008;8(3):207-209.
1.Schade R,Hlinak A. Egg yolk antibodies , state of the art and future prospects. ALTEX , 1996;13 (5) :5-9.
2.Klemperer F.Ueber natürliche Immunitat und ihre Verwerthung für die Immunisirungstherapie. A rchiv für Experimentelle Pathologie und Pharmakologie , 1893;31:356-82.
3.Suzuki N,Lee YC.Site-specific N-glycosylation of chicken serum IgG.Glyeobiology,2004;14(3):275-292.
4.FangYM,Kena W,Curran et a1.Polyclonal Gene Specific IgY Antibodies for Proteomies and Abundant Plasma Protein Depletion[M].New York:Frontiers of Biotechnology & Pharmaceuticals ,2003;4(5):134-141.
5.朱新产,王金宝,王宝维.鸡鸭鹅免疫球蛋白的研究进展[J].中国预防兽医学报,2003;25(6):503-508.
6.Shimizu M,Nagashima H,Sano K et al.Molecular stability of chicken and rabbit immunoglobulin G. Bioscience,Biotechnology and Biochemistry,1992; 56 (2):270-4.
7.Schmidt P,Wiedemann V,Kühlmann R et al.Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. II. In vitro studies on gastric and enteric digestion of egg yolk antibodies specific against pathogenic Escherichia coli strains. Zentralbl Veterinarmed B.1989 ;36 (8):619-28.
8.Wu C,Yang R,Zhou J,et a1.Production and characterisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase 1.Journal of Immunological Methods,2003;277(1-2):157-169.
9.Carlander D,Larsson A.Avian antibodies can eliminate interference due to complement activation in ELISA.Ups J Med Sci. 2001;106(3):189-95.
10.张小莺,郑礼,Rüdiger Schade1,et al.免疫鸡产生IgY抗体的技术[J] .中国药理学通报,2004;10:1102-1106.
11.Schwarzkopf C,Staak C,Behn I,Erhard M.Chicken egg yolk antibodies,production and application:IgY-technology[M],Berlin Heidelberg New York,2000;25-64.
12.Erhard MH,Ozpinar H,Bilal T,et al.The humoral immune response and the productivity of laying hens kept on the ground or in cages.Altern Lab Anim, 2000;28(5):699-705.
13.Hedlund GB,Hau J.Oral immunisation of chickens using choleratoxin B subunit and Softigen as adjuvants results in high antibody titre in the egg yolk.In vivo,2001;15(5):381-384.
14.Calzado EG,Marino EC,Chavez TS,et al.Extraction of a monospecific Coombs-reagent from chicken eggs.ALTEX,2003;20(1):21-25.
15.宋宏新,高栋.制备鸡卵黄免疫球蛋白的去脂方法研究进展[J].食品科学,2001;23(6):29-32.
16.Polson A,von Wechmar MB,van Regenmortel MH.Isolation of viral IgY antibodies from yolk of immunized hens.Immunol Commun,1980;9(5):475-493.
17.Polson A,Coetzer T,Kruger,et al.Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens.Immunol Invest,1985;14(4):323-327.
18.Poison A.Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure.Immunol Invest,1990,19(3):253-258.
19. Akita E M,Nakai S.Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.Immunol Methods,1993,160(2):207-14.
20.Verdoliva A,Basile G,Fassina G.Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand.Chromatogr B Biomed Sci Appl,2000;749(2):233-242.
21.王勇德,李航,赵国华,等.免疫蛋黄抗体的研究进展[J].中国食品学报,2005;5(1):99-102.
22.Hassl A,Aspock H. Purification of egg yolk immunogobulins. A two-step procedure using hydrophobic interaction chromatography and gel fitration [J].Immunol Method,1988;110(2):225-228.
23.魏英杰,丁金凤,赵勇,等.成人动脉和心脏cDNA文库的构建及新全长cDNAs的克隆.中国循环杂志,1999,14(6):369-371.
24.Zhao Y,Meng XM,Wei YJ,et a1.Cloning and characterization of a novel cardiac-specific kinase that interacts specifically with cardiac troponin I.Mol Med,2003;81(5):297-304.
25.尤昕,时娜,赵秀文,等.心肌特异表达基因p93的原核表达及鸡卵黄抗体的制备.中华检验学杂志,2005;28(10):1060-1063.
26.Brunda G,Sashidhar RB,Satin RK.Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin.Toxicon,2006;48(2):183-194.
27.de Almeida CM,Quintana-Flores VM,Medina-Acosta E,et al.Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli. Scand J Immunol,2003 ;57 (6) :573-582.
28.Lemamy GJ,Roger P,Mani JC,et al.High-affinity antibodies from hen's-egg yolks against human mannose-6-phosphate/insulin-like growth-factor-II receptor (M6P/IGFII-R):characterization and potential use in clinical cancer studies.Int J Cancer,1999;80(6):896-902.
29.Berthe ML,Esslimani Sahla M, Roger P,et al.Mannose-6-phosphate/insulin-like growth factor-II receptor expression levels during the progression from normal human mammary tissue to invasive breast carcinomas Eur J Cancer,2003 ; 39(5) :635-642.
30.Davalos-Pantoja L,Ortega-Vinuesa JL,Bastos-Gonzalez D,etal.A comparative study between the adsorption of IgY and IgG on latex particles.J Biomater Sci Polym Ed,2000;11(6):657-673.
31.Hatta H,Tsuda K,Ozeki M,et al. Passive immunization against dental plaque formation in humans:effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans. Caries Res,1997;31(4):268-274.
32.Shin J H,Yang M,Nam SW,et al.Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection. Clin Diagn Lab Immunol,2002;9(5):1061-1066.
33.Lee EN,Sunwoo HH,Menninen K,et al.In vitro studies of chicken egg yolk antibody (IgY)against Salmonella enteritidis and Salmonella typhimurium.Poultry Science,2002;81(5):632-641.
34.郭立君,李春晖,赵峰,等.抗轮状病毒IgY的研制.中国生物制品学杂志,2001;14(1):24—27.
35.Sarker SA,Casswall TH,Juneja LR,et al. Randomized, Placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea.Pediatr Gastroenterol Nutr,2001;3(1):19-25.
36.王玉梅,王敏文,张雪梅,等.抗流感病毒特异性IgY对小鼠的保护作用研究.生物学杂志,2004;21(4):21-22.
37.Yang J,Jin Z,Yu Q,et al. The selective recognition of antibody IgY for digestive system cancers. Chin J Biotechnol ,1997;13 (2):85-90.
38.Hensel T, Amberger VR,Schwab ME. A metalloproteaseactivity from C6 glioma cells inactivates the myelin-associated neurite growth inhibitors and can be neutralized by antibodies.Br J Cancer ,1998;78(12):1564-1572.
39.Suri A.Family of sperm associated antigens:relevance in spermeg interaction and immunocontraception[J].Soc Reprod Fertil Suppl.2007;63:433-443.
40.吕年青,黄宇烽,赵金然,等.鸡抗人精子卵黄抗体IgY的制备[J].中华男科学,2000;6(2):lll-ll3.
41.聂荣庆,吴东风,胡国柱,等.特异性IgY抗体对中性粒细胞吞噬功能的影响[J].中国兽医杂志,2004;40(12):23-26.
2.詹斌,肖树华,李铁华,等.钩虫流行现状及疫苗研制进展[J].中国寄生虫学与寄生虫病杂志,2000 ;18(3):182-185.
3.Brooker S,Bethony J,Hotez PJ.Hotez. Human Hookworm Infection in the 21st Century.J Adv Parasitol,2004;58:197-288.
4.胡锡敏,林绍雄,王善青,等.海南省人体钩虫感染情况现状调查分析[J].寄生虫与医学昆虫学报,2007;14(3):145-148.
5.谢红,田洪春,唐中玖,等.四川省钩虫等肠道线虫病2005-2006年监测数据分析[J].寄生虫病与感染性疾病,2008;6(1):21-24.
6.Hotez PJ. Hookworm disease in children.Pediatr Infect Dis ,1989;8(8):516-520.
7.杨毅.钩虫病所致上消化道大出血2例报告[J].江西医药,1999;33(4):236-237.
8.徐明琪.婴儿钩虫病62例临床分析[J].四川医学,2001;22(1):35.
9.罗秀亭,张平安,钱铃.钩虫病误诊32例[J].临床军医杂志,2006;34(3):371-372 .
10.杨松,钩虫病误诊支气管哮喘、慢性阻塞性肺疾病1例分析,医药世界,2009;11(7):343-344.
11.潘卫庆.寄生虫生物学研究与应用[M].北京:化学工业出版社,2007:266.
12.Albonico M,Smith PG,Ercole E,et a1.Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area. Trans R Soc Trop Med Hyg,1995;89(5):538-41.
13.Pritchard DI,Quinnell RJ,Slater AF,et al.Epidemiology and immunology of Necator americanus infection in a community in Papua New Guinea:humoral responses to excretory-secretory and cuticular collagen antigens.Parasitology,1990;100(2):317-326.
14.SorghoH,Bahgat M, Poda J N, et al. Serodiagnosis of Schistosoma mansoni infections in an endemic area of Burkina Faso: performance of several immunological tests with different parasite antigens. Acta Trop,2005;93(2):169-180.
15.Albonico M,Smith PG,Ercole E,et al. Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area.Trans R Soc Trop Med Hyg,1995;89(5):538-541.
16.Prichard R.Genetic variability following selection of Haemonchus contortus with anthelmintics .Trends Parasitol,2001;17(9):445-53.
17.Fincham JE, Markus MB, Adams VJ.Could control of soil-transmitted helminthic infection influence the HIV/AIDS pandemic.Acta Tropica,2003;86(2-3):315-33.
18.杨平生.咪唑类驱虫药致脱髓鞘脑病21例临床分析[J].医学临床研究,2004;21(7):748-750.
19.Alan D.T.Barrett,Lawrence Stanberry.Hookworm Infection. Vaccines for Biodefense and Emerging and Neglected Diseases[M],new york:Academic Press,2009:1365-78.
20.詹斌,肖树华,李铁华,等.钩虫流行现状及疫苗研制进展[J],中国寄生虫学与寄生虫病杂志,2000 ;18(3):182-185.
21.Suzuki N,Lee YC,Site-specific N-glycosylation of chicken serum IgG.Glyeobiology,2004;14(3):275-292.
22.FangYM,KenaW,Curran et a1.Polyclonal Gene Specific IgY Antibodies for Proteomies and Abundant Plasma Protein Depletion[M].New York:Frontiers of Biotechnology & Pharmaceuticals ,2003;4(5):134-141.
23.Schade R,Hlinak A.Egg yolk antibodies,state of the art and future prospects. ALTEX,1996;13(5):5-9.
24.Hatta H,Tsuda K,Ozeki M,et al.Passive immunlzation against dental plaque formation in humans:effect of a mouth rinse containing egg yolk antibodies (IgY)specific to streptccoccus mutarm.Caries Res,1997;31(4):268-274.
25.Sarker SA,Casswall TH,Juneja LR et al. Randomized,Placebo-controlled,clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children withrotavirus diarrhea.Pediatr Gastroenterol Nutr,2001;32(1):19-25.
26.聂荣庆,吴东风,胡国柱,等.特异性IgY抗体对中性粒细胞吞噬功能的影响[J].中国兽医杂志,2004;40(12):23-26.
27.Humphrey BD,Calvert CC,Klasing KC.Klasing The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos),Developmental and Comparative Immunology,2004;28(7-8):665–672.
28.Lee EN,Sunwoo HH,Menninen K,et al.In vitro studies of chicken egg yolk antibody (IgY) against Salmonella enteritidis and Salmonella typhimurium. Poultry Science,2002;81(5):632~641.
29.Akita EM,Nakai S.Comparison of four purification methods for the production of immunoglobulins from egs laid by hens immunized with an enterotoxigenic E.coli strain[J].J Immunol Methods,1993;163(1):141-142.
30.许正敏,李智山,孙莉,等.犬钩蚴固体培养基滤纸培养法[J].中国寄生虫学与寄生虫病杂志,2007;25(4):358.
31.Xiao Shuhua,Peter J,Hotez,etal.Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae .Parasitology International,2001;50(4):241-248.
32.SorghoH,Bahgat M, Poda J N, et al. Serodiagnosis of Schistosoma mansoni infections in an endemic area of Burkina Faso: performance of several immunological tests with different parasite antigens. Acta Trop,2005;93(2):169-180.
33.朱新产,王金宝,王宝维.鸡鸭鹅免疫球蛋白的研究进展[J].中国预防兽医学报,2003;25(6):503-508.
34.Bizhanov G,Vyshniauskis G.A comparison of three methods for extracting IgY from the egg yolk of hens immunized with Sendai virus.Vet Res Commun.2000;24(2) :103-13.
35.Mine Y,Kovacs-Nolan J.Chicken egg yolk antibodies as the rapeutics in enteric infectious disease:a review. J Med Food,2002,5 (3):159-169.
36.Davalos-Pantoja L,Ortega-Vinuesa JL,Bastos-Gonzalez D,etal.A comparative study between the adsorption of IgY and IgG on latex particles.J Biomater Sci Polym Ed ,2000 ,11(6) :657-673.
37.Shin J H ,Yang M ,Nam SW ,et al. Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection.Clin Diagn Lab Immu-nol ,2002 ,9 (5) :1061-1066.
38.Erhard MH,Ozpinar H,Bilal T,et al.The humoral immune response and the productivity of laying hens kept on the ground or in cages.Altern Lab Anim,2000;28(5):699-705.
39.Schade R,Staak C,Hendriksen C,et al.The production of avian(Egg yolk)antibodies:IgY.The report and recommendations of ECVAM workshop21.ATLA,1996;24(6):925-934.
40.常山,张雅萍,查晓霞,等.抗白色念珠菌鸡卵黄抗体(IgY)的研制[J].第三军医大学学报,2002;24(9):1026-1028.
41.Lee KA,Chang SK,Lee YJ et al .Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution.Biochemist ry and Molecular Biology,2002;35 (5):488-493.
42.Kricka LJ.Human anti-animal antibody interferences in immunological assays.Clin Chem,1999;45(7):942-956.
43.Lee EN,Sunwoo HH,Menninen K,et al.In vitro studies of chicken egg yolk antibody (IgY) against Salmonella enteritidis and Salmonella typhimurium.Poultry Science,2002;81(5):632-41.
44.Humphrey BD,Calvert CC,Klasing KC.The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos).Dev Comp Immunol,2004;28(7-8):665-72.
45.聂荣庆,吴东风,胡国柱,等.特异性IgY抗体对中性粒细胞吞噬功能的影响[J],中国兽医杂志,2004;40(12):23-25.
46.郭冠英,杨路.IgY牙膏对远缘链球菌体外黏附及超微结构的影响[J].中国药物与临床,2008;8(3):207-209.
1.Schade R,Hlinak A. Egg yolk antibodies , state of the art and future prospects. ALTEX , 1996;13 (5) :5-9.
2.Klemperer F.Ueber natürliche Immunitat und ihre Verwerthung für die Immunisirungstherapie. A rchiv für Experimentelle Pathologie und Pharmakologie , 1893;31:356-82.
3.Suzuki N,Lee YC.Site-specific N-glycosylation of chicken serum IgG.Glyeobiology,2004;14(3):275-292.
4.FangYM,Kena W,Curran et a1.Polyclonal Gene Specific IgY Antibodies for Proteomies and Abundant Plasma Protein Depletion[M].New York:Frontiers of Biotechnology & Pharmaceuticals ,2003;4(5):134-141.
5.朱新产,王金宝,王宝维.鸡鸭鹅免疫球蛋白的研究进展[J].中国预防兽医学报,2003;25(6):503-508.
6.Shimizu M,Nagashima H,Sano K et al.Molecular stability of chicken and rabbit immunoglobulin G. Bioscience,Biotechnology and Biochemistry,1992; 56 (2):270-4.
7.Schmidt P,Wiedemann V,Kühlmann R et al.Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. II. In vitro studies on gastric and enteric digestion of egg yolk antibodies specific against pathogenic Escherichia coli strains. Zentralbl Veterinarmed B.1989 ;36 (8):619-28.
8.Wu C,Yang R,Zhou J,et a1.Production and characterisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase 1.Journal of Immunological Methods,2003;277(1-2):157-169.
9.Carlander D,Larsson A.Avian antibodies can eliminate interference due to complement activation in ELISA.Ups J Med Sci. 2001;106(3):189-95.
10.张小莺,郑礼,Rüdiger Schade1,et al.免疫鸡产生IgY抗体的技术[J] .中国药理学通报,2004;10:1102-1106.
11.Schwarzkopf C,Staak C,Behn I,Erhard M.Chicken egg yolk antibodies,production and application:IgY-technology[M],Berlin Heidelberg New York,2000;25-64.
12.Erhard MH,Ozpinar H,Bilal T,et al.The humoral immune response and the productivity of laying hens kept on the ground or in cages.Altern Lab Anim, 2000;28(5):699-705.
13.Hedlund GB,Hau J.Oral immunisation of chickens using choleratoxin B subunit and Softigen as adjuvants results in high antibody titre in the egg yolk.In vivo,2001;15(5):381-384.
14.Calzado EG,Marino EC,Chavez TS,et al.Extraction of a monospecific Coombs-reagent from chicken eggs.ALTEX,2003;20(1):21-25.
15.宋宏新,高栋.制备鸡卵黄免疫球蛋白的去脂方法研究进展[J].食品科学,2001;23(6):29-32.
16.Polson A,von Wechmar MB,van Regenmortel MH.Isolation of viral IgY antibodies from yolk of immunized hens.Immunol Commun,1980;9(5):475-493.
17.Polson A,Coetzer T,Kruger,et al.Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens.Immunol Invest,1985;14(4):323-327.
18.Poison A.Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure.Immunol Invest,1990,19(3):253-258.
19. Akita E M,Nakai S.Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.Immunol Methods,1993,160(2):207-14.
20.Verdoliva A,Basile G,Fassina G.Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand.Chromatogr B Biomed Sci Appl,2000;749(2):233-242.
21.王勇德,李航,赵国华,等.免疫蛋黄抗体的研究进展[J].中国食品学报,2005;5(1):99-102.
22.Hassl A,Aspock H. Purification of egg yolk immunogobulins. A two-step procedure using hydrophobic interaction chromatography and gel fitration [J].Immunol Method,1988;110(2):225-228.
23.魏英杰,丁金凤,赵勇,等.成人动脉和心脏cDNA文库的构建及新全长cDNAs的克隆.中国循环杂志,1999,14(6):369-371.
24.Zhao Y,Meng XM,Wei YJ,et a1.Cloning and characterization of a novel cardiac-specific kinase that interacts specifically with cardiac troponin I.Mol Med,2003;81(5):297-304.
25.尤昕,时娜,赵秀文,等.心肌特异表达基因p93的原核表达及鸡卵黄抗体的制备.中华检验学杂志,2005;28(10):1060-1063.
26.Brunda G,Sashidhar RB,Satin RK.Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin.Toxicon,2006;48(2):183-194.
27.de Almeida CM,Quintana-Flores VM,Medina-Acosta E,et al.Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli. Scand J Immunol,2003 ;57 (6) :573-582.
28.Lemamy GJ,Roger P,Mani JC,et al.High-affinity antibodies from hen's-egg yolks against human mannose-6-phosphate/insulin-like growth-factor-II receptor (M6P/IGFII-R):characterization and potential use in clinical cancer studies.Int J Cancer,1999;80(6):896-902.
29.Berthe ML,Esslimani Sahla M, Roger P,et al.Mannose-6-phosphate/insulin-like growth factor-II receptor expression levels during the progression from normal human mammary tissue to invasive breast carcinomas Eur J Cancer,2003 ; 39(5) :635-642.
30.Davalos-Pantoja L,Ortega-Vinuesa JL,Bastos-Gonzalez D,etal.A comparative study between the adsorption of IgY and IgG on latex particles.J Biomater Sci Polym Ed,2000;11(6):657-673.
31.Hatta H,Tsuda K,Ozeki M,et al. Passive immunization against dental plaque formation in humans:effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans. Caries Res,1997;31(4):268-274.
32.Shin J H,Yang M,Nam SW,et al.Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection. Clin Diagn Lab Immunol,2002;9(5):1061-1066.
33.Lee EN,Sunwoo HH,Menninen K,et al.In vitro studies of chicken egg yolk antibody (IgY)against Salmonella enteritidis and Salmonella typhimurium.Poultry Science,2002;81(5):632-641.
34.郭立君,李春晖,赵峰,等.抗轮状病毒IgY的研制.中国生物制品学杂志,2001;14(1):24—27.
35.Sarker SA,Casswall TH,Juneja LR,et al. Randomized, Placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea.Pediatr Gastroenterol Nutr,2001;3(1):19-25.
36.王玉梅,王敏文,张雪梅,等.抗流感病毒特异性IgY对小鼠的保护作用研究.生物学杂志,2004;21(4):21-22.
37.Yang J,Jin Z,Yu Q,et al. The selective recognition of antibody IgY for digestive system cancers. Chin J Biotechnol ,1997;13 (2):85-90.
38.Hensel T, Amberger VR,Schwab ME. A metalloproteaseactivity from C6 glioma cells inactivates the myelin-associated neurite growth inhibitors and can be neutralized by antibodies.Br J Cancer ,1998;78(12):1564-1572.
39.Suri A.Family of sperm associated antigens:relevance in spermeg interaction and immunocontraception[J].Soc Reprod Fertil Suppl.2007;63:433-443.
40.吕年青,黄宇烽,赵金然,等.鸡抗人精子卵黄抗体IgY的制备[J].中华男科学,2000;6(2):lll-ll3.
41.聂荣庆,吴东风,胡国柱,等.特异性IgY抗体对中性粒细胞吞噬功能的影响[J].中国兽医杂志,2004;40(12):23-26.