【作者】 彭伏虎；

【导师】 毕丁仁；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 禽流感（AI）与新城疫（ND）是世界公认的最重要的两大禽类传染病,可导致禽类的大量死亡或生产性能的急剧下降,给世界养禽业造成巨大的经济损失,同时由于禽流感病毒（AIV）可以感染人,引起人发病甚至死亡,给人类的公共卫生安全造成了极大的威胁。目前,对于AI和ND的诊断主要有病毒分离、血凝和血凝抑制、ELISA、RT-PCR等,但由于耗时冗长或需要特殊的仪器设备及技术人员,难以满足现场、适时快速诊断的要求。胶体金免疫层析方法是以条状纤维层析材料为固相载体,通过毛细管作用原理,以胶体金为示踪物来显示结果的一种新型的检测方法。这种方法具有操作简单、快捷,分析结果清楚、易于判断,且不需要任何仪器、成本低廉等优点,非常适用于现场检测及自动化检测分析。因此,在医学快速检测领域得到了迅猛发展,目前已逐渐应用于动物医学领域的快速检测。本研究针对目前禽类呼吸道疾病病原检测方法中存在的问题,运用胶体金免疫层析技术,采用双抗体夹心法原理,建立了两种禽类主要呼吸道病原的快速检测技术,取得了如下研究成果。1.鸡抗AIV、鸡抗NDV和山羊抗鼠多克隆抗体的制备及纯化用通过鸡胚传代,经超离浓缩后的AIV（H9N2）和NDV免疫健康鸡,同时,以纯化的小鼠IgG免疫山羊,制备了鸡抗AIV、鸡抗NDV、羊抗鼠高免血清。对所得高免血清分别采用饱和硫酸铵粗提和G-200过柱纯化,最后得到了纯化的鸡抗AIV、鸡抗NDV和羊抗鼠抗体。2.单克隆抗体的制备与纯化利用纯化的AIV融合表达蛋白GST-NP和NDV免疫BALB/c小鼠,制备免疫脾细胞,经过细胞融合、筛选和多次克隆分别获得5株针对AIV-NP,5株针对NDV-HN的能稳定分泌单克隆抗体的杂交瘤细胞。同时对实验室制备和保存的禽流感H55E8和H94C4单克隆细胞株成功进行了复苏。经小鼠腹腔接种获得的单抗效价均较高,5株针对AIV-NP的ELISA效价均在1:3.2×10⁴以上;5株针对NDV-HN的ELISA效价均在1:1.6×10⁴以上,HI效价均在1:2¹⁰以上;禽流感H55E8和H94C4的腹水HI效价分别为1:2¹⁴和1:2¹⁶。对其中效价高的几株进行了纯化,获得了高纯度的单克隆抗体。3.胶体金及金标抗体的制备采用柠檬酸三钠还原法,成功确立了20nm胶体金的制备条件;并利用制备好的胶体金,对前述制备的抗禽流感核蛋白单克隆抗体（AIV-NP5F4）、抗禽流感H9亚型血凝素单克隆抗体（H94C4）、抗禽流感H5亚型血凝素单克隆抗体（H55E8）、抗新城疫病毒血凝素神经氨酸酶单克隆抗体（NDV-HN2I3）进行了胶体金标记条件的摸索,成功确立了单克隆抗体的胶体金标记条件:最适pH值为8.0,最适标记量为12μg/mL胶体金,并制备了相应的金标抗体。4.H5、H9亚型禽流感病毒免疫层析快速检测及鉴别诊断试纸条的研制以胶体金标记禽流感H5、H9亚型血凝素单克隆抗体作为示踪物,用固定在检测线上的鸡抗AIV IgG作为捕获抗体,采用双抗体夹心法,分别成功地建立了H5、H9亚型AIV免疫层析快速检测试纸条。用制成的H9亚型AIV试纸条检测已知H9N2亚型AIV尿囊液和禽流感H9亚型标准血凝抗原时,检测线出现明显的紫红色条带,而阴性对照、其它AIV标准血凝抗原以及多种禽类病毒包括EDS-76V、NDV、IBV、ILTV,IBDV等均呈阴性反应;用制成的H5亚型AIV试纸条检测已知禽流感H5亚型标准血凝抗原时,检测线出现明显的紫红色条带,而阴性对照、其它AIV标准血凝抗原以及多种禽类病毒包括EDS-76V、NDV、IBV、ILTV,IBDV等均呈阴性反应。效价测定的结果表明,H9亚型AIV检测试纸条的灵敏度可以达到0.25个血凝单位,比血凝试验高4倍以上,H5亚型AIV检测试纸条的灵敏度与HA试验相当。在此基础上,本研究用金标记抗禽流感病毒核蛋白单克隆抗体作为示踪物,在两条检测线上分别包被禽流感H9亚型血凝素单克隆抗体H94C4、禽流感H5亚型血凝素单克隆抗体H55E8作为捕获抗体,建立了H5、H9亚型AIV快速鉴别诊断试纸条。实验结果证明,本方法特异性强,灵敏度高,且操作简便、结果判定直观,可以用于H5、H9亚型AIV的快速检测及鉴别诊断。5.禽流感和新城疫病毒免疫层析快速检测及鉴别诊断试纸条的研制本研究以胶体金标记抗AIV-NP单克隆抗体作为示踪物,用固定在检测线上的鸡抗AIV IgG作为捕获抗体,采用双抗体夹心法,成功地建立了AIV胶体金快速检测试纸条。用制成的试纸条检测已知H5、H7、H9亚型禽流感病毒标准血凝抗原时,检测线出现明显的紫红色条带,而阴性对照、其它禽类病毒包括EDS-76V、NDV、IBV、ILTV,IBDV等均呈阴性反应;效价测定的结果表明,试纸条法的灵敏度比血凝试验高4倍以上。以胶体金标记抗NDV-HN单克隆抗体作为示踪物,用固定在检测线上的鸡抗NDV IgG捕获抗体,采用双抗体夹心法,成功地建立了NDV胶体金快速检测试纸条。用制成的试纸条检测已知NDV时,检测线出现明显的紫红色条带,而阴性对照,H5、H7、H9亚型禽流感病毒标准血凝抗原以及其它禽类病毒包括EDS-76V、IBV、ILTV,IBDV等均呈阴性反应;效价测定的结果表明,试纸条法的灵敏度比血凝实验至少高4倍。在此基础上,采用一膜包被多个抗体的方法,用混合后的金标记抗AIV-NP单克隆抗体和胶体金标记抗NDV-HN单克隆抗体作为示踪物,建立了AIV和NDV快速鉴别诊断试纸条。实验结果证明,本方法特异性强,灵敏度高,且操作简便、结果判定直观,可以用于AIV和NDV的检测与鉴别诊断。6.禽流感病毒通用型及H9亚型快速检测试剂盒的研制与应用用建立的快速检测方法成功研制出禽流感病毒快速检测试剂盒和H9亚型禽流感病毒快速检测试剂盒。分别对两种试剂盒进行了特异性、敏感性、重复性及保存期的测定。结果表明,在4℃能保存12个月,室温能保存6个月而特异性和敏感性没有任何变化,而且特异性强、敏感性高、重复性好,操作简单、方便,能快速、准确地检测出样品中是否带有禽流感病毒或H9亚型禽流感病毒抗原。对试剂盒的验证结果表明,两种试剂盒均能在感染三天之内检出禽流感病毒,真正达到了早期检测的要求。临床采集311份样品,用H9亚型禽流感病毒快速检测试剂盒及禽流感病毒快速检测试剂盒进行检测,检出率分别为35%和39.2%,与本室早期研制的禽流感病原ELISA诊断试剂盒的符合率分别为92%和100%,对试剂盒检测样品的病毒分离鉴定证明无一漏检。更多还原

【Abstract】 Avian influenza（AI） and New castle disease are the two capital contagious disease in birds caused by avian influenza virus（AIV） and new castle disease virus（NDV） respectively.Both avian influenza and new castle disease can cause tremendous economic losses in poultry industry and since avian influenza virus can infect human beings and cause disease even death,which become a threat for humans and raise great public health concern.Various laboratory methods are currently available for the detection and surveillance of AIV,and include virus isolation,hemagglutination（HA） assay, hemagglutination inhibition（HI） test,enzyme linked immunoabsorbent assay（ELISA） and RT-PCR.However,these assays are laborious and time consuming,difficult to incorporate into automated procedure,and require laboratory operation,skilled technicians,and special equipment/facilities,are limited in spot,real time and rapid application.Immunochromatographic assay is a new immunochromatographic technique in which a cellulose membrane is used as the carrier,and a colloidal gold-labeled antigen or antibody is used as the tracer.It is simple,rapid and cheap,and the result is clear and easy to analyze without any equipment/facilities and skilled technicians.Since it is very suitable for spot,rapid detection and automatic detection,the method has been widely used for rapid detection in human medicine,and more recently,it has been used for rapid detection in veterinary medical analysis.Facing the shortage of the present methods used in detecting pathogens of respiratory disease in poultry,a technology system for rapid detecting pathogens of respiratory disease in poultry was drawn up using the principle and technology of colloidal gold immunochromatographic assay.This study focused on developing and evaluating the method for rapid detection of avian influenza virus and new castle disease virus and the results were reported as follows.1.Preparation of polyclonal antibodies of chicken anti-AIV,chicken anti-NDV and goat anti-mouse IgGAIV（H9N2） and NDV were inoculated into 9 to 10-day embryonating chicken eggs by the allantoic route respectively,then allantoic fluid was harvested and enriched by centrifuging at 30,000r/m.Chickens were immunized with prepared AIV and NDV emulsified with Frund’s adjuvant,at the same time,goats were immunized with purified mouse IgG emulsified with Frund’s adjuvant.Sera of chicken anti-AIV,chicken anti-NDV and goat anti-mouse IgG were acquired after several inoculations,and the titers of AGID reach 1:64.The depurated antibodies were obtained after these sera were purified by（NH₄）₂SO₄ precipitation and G-200 respectively.2.Preparation monoclonal antibodies used in this studyAfter immunization of BALB/C mice with the purified NDV and GST-NP from AIV respectively,the stimulated splenocytes were routinely fused with SP2/0 myeloma cells to produce hybridomas.After three cycles of cloning,5 stable hybridoma clones producing monoclonal antibodies to AIV and 5 to NDV were obtained.At the same time, Hybridoma cells H55E8 and H94C4 were recovered which were previously prepared in our lab.All the Hybridoma cells were cultured in RPMI-1640 with 5%FCS,harvested and injected into nude mice,and then mouse ascetic fluids were harvested 10 days later. The ELISA titers of 5 monoclonal antibodies for AIV-NP were 1:32000～1:64000 and 5 monoclonal antibodies for NDV-NH were 1:16000～1:64000 with the HI titers of 1:1024～1:16384.The HI titer of monoclonal antibody H55E8 and H94C4 were 1:16384 and 1:65536 respectively.Selected monoclonal antibodies with higher titers were purified from mouse ascetic fluids using sequential precipitation with caprylic acid and ammonium sulfate.3.Preparation of colloidal and immuno-colloidal,goldAfter the preparing conditions were established,colloidal gold of 20nm was prepared with reducing reaction using trisodium citrate.Studies were progressed on the optimal conditions about preparing the colloidal gold conjugates with monoclonal antibodies previously prepared,such as monoclonal antibody 5F4 against neucleoprotein of AIV,monoclonal antibody H94C4 against hemagglutinin of H9AIV,monoclonal antibody H55E8 against hemagglutinin of H5AIV and monoclonal antibody 213 against hemagglutinin-neuramidinase of NDV.After the optimal conditions were developed（the optimal pH is 8.0,the optimal mount of monoclonal antibody is 12μg/mL）,colloidal gold-5F4 conjugate,colloidal gold-H94C4 conjugate,colloidal gold- H55E8 conjugate and colloidal gold-213 conjugate were prepared respectively according to the method previously described.4.Development of immunochromatographic strips for rapid detection and differential diagnosis of HS,H9 subtype of avian influenza virusesTwo immunochromatographic strips were developed respectively for rapid detection of the H5,H9 subtype of avian influenza viruses in poultry with sandwich method.The monoclonal antibody 4C4 for H9AIV hemagglutinin,5E8 for H5AIV were labeled with colloidal gold as the detection reagent respectively,and the chicken anti-AIV IgG was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane.To determine the specificity of the immunochromatographic strip for the detection of H9AIV,allantoic fluids harvested from H9N2 infected eggs at 64 units of HA,allantoic fluids from unmanipulated eggs, together with standard antigens of H5,H7,or H9AIV and other viruses,were characterized using the immunochromatographic strips simultaneously.Clearly,while all of the samples tested showed one strong band on the control line,only allantoic fluid harvested from H9N2 infected eggs and the standard H9 AIV antigen displayed an additional band on the test line on the immunochromatographic strips.This suggests that the immunochromatographic strip can specifically detect H9AIV,but not other tested AIVs,NDV,IBV,IBDV,ILTV,and FAV,common infectious viruses in poultry.To determine the specificity of the immunochromatographic strip for the detection of H5AIV,allantoic fluids from unmanipulated eggs,together with standard antigens of H5, H7,or H9AIV and other viruses,were characterized using the immunochromatographic strips simultaneously.Clearly,while all of the samples tested showed one strong band on the control line,only the standard H5AIV antigen displayed an additional band on the test line on the immunochromatographic strips.This suggests that the immunochromatographic strip can specifically detect H5AIV,but not other tested AIVs, or other common infectious viruses in poultry.In comparison with the hemagglutination （HA） and hemagglutination inhibition（HI） test,the sensitivity of the immunochromatographic strip for the detection of H9AIV reached a level of 0.25 units of HA antigens,approximately 4 times higher than HA test,while the immunochromatographic strip for the detection of H5AIV only reached the HA test level. At the same time,another immunochromatographic strip was developed for the differential diagnosis of H5 and H9 subtype of avian influenza virus.The monoclonal antibody 5F4 for neucleoprotein of AIV was labeled with colloidal gold as a detection reagent,and the monoclonal antibody 4C4 for H9AIV and monoclonal antibody 5E8 for H5AIV were blotted on the nitrocellulose membrane with 5mm wide width as test line 1 and test line 2 respectively.Results suggested that the strips were easy to operate and can be applied in rapid detecting and differential diagnosing H5,H9 subtype of AIV with high specificity and sensitivity within 10 minute.5.Development of immunochromatographic strips for rapid detection and differential diagnosis of avian influenza virus and New castle disease virusAn immunochromatographic strip was developed for the detection of AIV in poultry with sandwich method.The monoclonal antibody 5F4 for neucleoprotein of AIV was labeled with colloidal gold as a detection reagent,and the chicken anti-AIV IgG was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane.To determine the specificity of the immunochromatographic strip,standard antigens of H5,H7,or H9AIV and other viruses were characterized using the immunochromatographic strips simultaneously.Clearly, while all of the samples tested showed one strong band on the control line,only the standard H5,H7 and H9 AIV antigen displayed an additional band on the test line on the immunochromatographic strips.This suggests that the immunochromatographic strip can specifically detect AIV,but not other tested virus common infected in poultry,such as NDV,IBV,IBDV,ILTV,and FAV,.In comparison with the hemagglutination（HA） and hemagglutination inhibition（HI） test,the sensitivity of the immunochromatographic strip reached a level of 0.25 units of HA antigens,approximately 4 times higher than HA test.At the same time,another immunochromatographic strip was developed for the detection of NDV in poultry with sandwich method.The monoclonal antibody 213 for hemagglutinin neuraminidase of NDV was labeled with colloidal gold as a detection reagent,and the chicken anti-NDV IgG was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane.One strong band showed on control line when the standard H5,H7,H9 AIV antigens and other virus common infected in poultry were tested by the strip,while an additional band showed on test line when detecting NDV.Results suggested that the strip can specifically detect NDV with high sensitivity at 4 times higher than HA.Based on the previous work,an immunochromatographic strip was developed for the differential diagnosis of NDV in poultry.The colloidal gold-5F4 conjugate and colloidal gold-213 were mixed as the detection reagent,and the chicken anti-NDV IgG and chicken anti-AIV IgG were blotted on the nitrocellulose membrane with 5mm wide width as test line 1 and test line 2 respectively.Results suggested that the strip can be applied in differential diagnosing NDV and AIV with high specificity and sensitivity within 10 minute.6.Preparation and application of kits for rapid detection of general type and H9 subtype avian influenza virusTwo kits were developed for rapid detection of avian influenza virus and H9 subtype avian influenza virus based on rapid detecting immunochromatographic assay previously developed.According to the method described above,experiments were progressed to evaluate the specificity,sensitivity,reproducibility and stability.Storage of the kits at room temperature for six months or at 4℃for 12 months did not change their sensitivity and specificity.Evaluation of the kits on experimental tracheal and cloacal swab samples collected from HgN2-infected chickens revealed that both of the two rapid test kits detected the HgN2 viruses on day 3 post-inoculation,earlier than the appearance of clinical symptoms.Application of the kits for detection of AIV and H9AIV for analysis of 311 samples collected from potentially infected chickens,the positive ratio was 39.2%and 35%with a coincidence at 100%and 92%respectively with the ELISA kits previously prepared in our lab.Further characterization samples randomly selected showed that no single sample was false positive or negative,as determined by the standard virus isolation and HI assays.Therefore,the two rapid test kits for the detection of AIV and H9AIV both have high specificity,sensitivity and stability.This,together with the advantages of rapid detection and easy operation without requirement for special skills and equipments,makes it suitable for the on-site detection of AIV and differentiation of H9AIV from other viruses in poultry.更多还原