犬细小病毒血凝抑制抗体效价胶体金免疫层析检测方法的建立及应用

试验旨在利用胶体金免疫层析技术建立快速检测犬血清中犬细小病毒（canine parvo virus, CPV）血凝抑制（haemagglutination inhibition, HI）抗体效价的方法,用于CPV疫苗免疫效果评价。采用双抗体夹心法,以抗CPV血凝相关抗原的单克隆抗体制备CPV抗原检测试纸条;将犬血清进行不同比例系列稀释后,分别与定量CPV抗原充分反应,滴入CPV胶体金试纸条,根据试纸条检测线（test line,T线）消失时的血清最高稀释倍数判断血清中CPV抗体的HI效价;用此方法检测86份犬血清样品,并与传统血凝抑制试验方法进行分析比较。结果显示,成功制备CPV抗原检测试纸条,确定了试纸条检测犬血清CPV-HI效价的反应条件和结果判定标准。结果表明,在检测不同稀释倍数犬血清反应后的CPV抗原时,能使试纸条T线消失时的血清最高稀释倍数与HI效价具有正相关性,犬血清最高稀释倍数乘以4即为HI效价;两种方法的符合率达90.7%。本试验初步建立了胶体金试纸条检测CPV血凝抑制效价的方法,为检测CPV-HI效价提供了一种操作简单、快速的试验方法,可用于CPV疫苗免疫效果评价。     

犬瘟热病毒单克隆抗体的研制及胶体金试纸条检测方法的建立

本研究利用原核表达、纯化的犬瘟热病毒(Canine distemper virus,CDV)N 蛋白免疫BALB/C小鼠,成功筛选到两株分泌CDV N 蛋白单克隆抗体的杂交瘤细胞株,分别命名为D3、D6。2株杂交瘤细胞分泌单克隆抗体敏感性、特异性良好,能识别不同来源的犬瘟热病毒株,单抗亚类均为IgG1。以D3作为金标抗体,D6作为检测抗体,兔抗鼠IgG作为质控线抗体,制备犬瘟热病毒胶体金检测试纸条,检测犬瘟热病毒敏感性为1000TCID50,能有效检测区分犬瘟热病毒、犬细小病毒(Canine parvovirus,CPV)、犬腺病毒2型(Canine adenovirus type 2,CAV2)、狂犬病病毒(Rabies virus,RV)。综上,本研究建立的胶体金试纸条检测方法,灵敏度高、特异性强,适用于犬瘟热病毒临床快速诊断。     

犬狂犬病毒抗体胶体金检测试纸卡的研制

为快速检测犬狂犬病毒IgG抗体,应用狂犬病毒G蛋白作为捕获抗原,鼠抗狂犬病毒G蛋白单克隆抗体和羊抗犬IgG分别作为胶体金标记物和诊断抗体,羊抗鼠IgG作为质控线,制备胶体金免疫层析抗体检测试纸。该试纸卡具有良好的特异性、敏感性、重复性和稳定性,与快速荧光灶抑制实验检测值对比总符合率为82.1%。研究显示,本试纸卡能够快速、准确检测犬狂犬病毒IgG抗体,可为临床诊断和宠物犬狂犬疫苗免疫效果的评价提供参考。     

猪细小病毒血凝抑制试验抗原、阳性血清与阴性血清的制备与鉴定

猪细小病毒（Porcine Parvovirus,PPV）血凝抑制试验抗原、阳性血清和阴性血清在猪细小病毒制品的效力检验中不可或缺,对猪细小病毒相关生物制品的质量控制至关重要。试验采用PPV 7909株病毒同步接种PK-15细胞制备血凝抑制试验抗原,用制备的抗原乳化后免疫豚鼠制备阳性血清,同时用未免疫的阴性豚鼠制备阴性血清。对抗原、阳性血清和阴性血清进行鉴定,结果表明,制备的PPV血凝抑制试验抗原HA效价达1:512;阳性血清HI效价达1:1024;阴性血清HI效价＜1:8,且特异性均良好。利用制备的血凝抑制试验抗原、阳性血清与不同PPV疫苗株的灭活抗原、阳性血清进行交叉反应试验,结果表明,制备的抗原与阳性血清具备良好的血清学交叉反应性,可用于PPV制品的统一评价。     

半定量检测新城疫抗体胶体金试纸条的研制

为建立半定量快速检测新城疫抗体方法，本研究利用柠檬酸钠还原氯金酸，形成胶体金颗粒，应用直径20nm的胶体金颗粒标记新城疫抗原，制作了半定量快速测定新城疫抗体的免疫胶体金试纸条。用试纸条检测60份样品，检测结果与血凝抑制试验（HI）法测得结果的符合率为93．8％，最小检测HI滴度为410g2，与其他病毒的阳性血清没有交叉反应，且室温下有效保存期为3个月。试纸条应用方便、快速，适合临床推广以及流行病学调查。     

Duration of serologic response to five viral antigens in dogs

OBJECTIVE: To determine whether vaccinated dogs either remained seropositive or responded serologically to revaccination for 5 key viral antigens after extended periods since their last vaccination. DESIGN: Serologic survey. ANIMALS: 322 healthy client-owned dogs. PROCEDURE: Dogs were > or = 2 years old and vaccinated against canine distemper virus (CDV), canine adenovirus-1 (CAV-1), canine adenovirus-2 (CAV-2), canine parainfluenza virus (CPIV), and canine parvovirus (CPV). On day 0, dogs were revaccinated with a vaccine from the same vaccine line as they had historically received. Antibody titers were measured in sera collected at day 0 (prevaccination titer) and 5 to 7 days later (postvaccination titer). Dogs were considered to have responded serologically if they had a day-0 serum neutralization titer to CDV > or = 1:32; a serum neutralization titer to CAV-1, CAV-2, or CPIV > or = 1:16; a hemagglutination inhibition titer to CPV > or = 1:80; or a > or = 4-fold increase in antibody titer after revaccination. RESULTS: The percentage of dogs that had titers at or greater than the threshold values or responded to revaccination with a > or = 4-fold increase in titer was 98.1% for CDV, 98.4% for CAV-1, 99.0% for CAV-2, 100% for CPIV, and 98.1% for CPV. CONCLUSIONS AND CLINICAL RELEVANCE: In most dogs, vaccination induced a response that lasted up to and beyond 48 months for all 5 antigens. Although not equivalent to challenge-of-immunity studies as a demonstration of efficacy, results suggest that revaccination with the same vaccine provides adequate protection even when given less frequently than the traditional 1-year interval. The study provides valuable information for clinicians to help determine appropriate revaccination intervals.     

免疫层析试纸条(ICS)检测禽流感抗体

以纯化的禽流感病毒（avian influenza virus,AIV）核蛋白作为捕捉抗原，金标兔抗鸡抗体作为金标二抗，建立检测禽流感血清抗体的胶体金免疫层析试纸条（immunochromatographic strip, ICS）。对标准阳性血清不同滴度进行检测，ICS试验的敏感性高于琼脂扩散试验（AGP）。特异性试验证实，禽流感的ICS试验与新城疫（ND）、传染性支气管炎（IBV）、鸡传染性法氏囊病（IBD）及减蛋综合症（EDS-76）均无交叉反应。同时，用ICS试验和HI试验检测105份鸡血清，两者具有较好的符合性。结果认为：禽流感ICS具有快速简便、特异、灵敏等优点。     

间接ELISA检测禽流感抗体方法的建立

应用醛化的鸡红细胞经吸附释放方法获得纯化的禽流感病毒 (AIV) ,经 triton X-1 0 0处理并反复冻融制备禽流感 EL ISA抗原。应用抗鸡 Ig轻链单抗 1 B7辣根过氧化物酶结合物建立了定量检测禽流感抗体水平的 EL ISA方法。确立了将鸡血清 1 0 0倍稀释监测 EL ISA效价(ET)的回归方程 y=0 .77452 x 0 .8793 5(r=0 .9466) ,可用于定量测定。血凝抑制 (HI)抗体与 EL ISA方法的比较试验表明 ,EL ISA法比血凝抑制试验敏感 1 .5倍以上     

犬轮状病毒单克隆抗体制备及胶体金试纸条检测方法的建立

【目的】 研发犬轮状病毒（Canine ratavirus,CRV）免疫学诊断试剂,制备并鉴定CRV特异性单克隆抗体,建立可用于检测CRV的胶体金免疫层析法。【方法】 以CRV临床分离株SL006株为免疫原免疫BALB/c雌性小鼠,用杂交瘤细胞法进行细胞融合,通过间接免疫荧光法（IFA）筛选可稳定分泌CRV单克隆抗体的杂交瘤细胞,并制备腹水,亲和层析法进行纯化获得单克隆抗体。对获得的单克隆抗体进行鉴定,经条件优化建立胶体金试纸条检测方法,对试纸条的灵敏度、特异性、重复性和应用情况进行评价。【结果】 获得了4株杂交瘤细胞,分别命名为2C12、4A5、1H3、5F3,IFA效价分别为1：6 400、1：12 800、1：1 600和1：3 200;重链亚类分别为IgG2b、IgG2a、IgG1和IgG2a,轻链亚类均为kappa;制备的胶体金试纸条检测线包被单克隆抗体4A5,对照线包被羊抗鼠IgG,金标垫包被胶体金标记的单克隆抗体2C12,对CRV病毒液的检测灵敏度为10^4.2 TCID₅₀/mL,检测犬源其他病毒犬细小病毒（Canine parvovirus,CPV）、犬副流感病毒（Canine parainfluenza virus,CPIV）、犬腺病毒Ⅰ型（Canine adenovirus-Ⅰ,CAV-Ⅰ）、CAV-Ⅱ、犬瘟热病毒（Canine distemper virus,CDV）病毒液及CRV阴性肛拭子和阴性粪便均为阴性;批内和批间重复性良好;利用制备的胶体金试纸条和RT-PCR方法对47份样品进行检测比较,两者符合率为93.6%。【结论】 本研究建立的CRV胶体金试纸条检测方法具有良好的敏感性、特异性、重复性,与RT-PCR方法符合率较高,可为CRV的临床快速诊断提供有效的检测方法。     

口蹄疫病毒感染与免疫鉴别诊断及免疫评价二联试纸的优化

【目的】建立口蹄疫病毒(FMDV)感染与免疫鉴别诊断及免疫评价二联试纸稳定的生产工艺,促进其产业化生产及临床应用。【方法】本研究以胶体金免疫探针结合猪IgG,FMDV结构蛋白(SP)及非结构蛋白(NSP)的表位多肽偶联载体蛋白制备人工抗原,设置两条检测线精准拦截抗体的检测模式,以试纸条特异性及敏感性为评价指标,对胶体金免疫探针用蛋白、试纸拦截用多肽抗原、检测线位置及喷膜缓冲液、金标蛋白保存液、样品垫缓冲液以及样品稀释液等参数进行优化。采用优化后的试纸检测266份田间猪血清样品,并与口蹄疫O型抗体液相阻断ELISA检测试剂盒(LPB ELISA)和3ABC阻断ELISA抗体检测试剂盒(3ABC ELISA)检测结果进行对比,计算该试纸与商品ELISA试剂盒的符合率。【结果】经优化后,口蹄疫感染与免疫鉴别诊断及免疫评价二联试纸生产工艺参数如下:以胶体金标记金黄色葡萄球菌A蛋白(SPA)为免疫探针;选择混合多肽的形式设置拦截线,以非结构蛋白多肽(BSA-NSPs)喷涂T1线,结构蛋白多肽(BSA-SPs)喷涂T2线,以0.1 mol/L Tris-HCl为喷膜缓冲液;以ddH₂O含15 mg/mL BSA、13.25 mg/mL Na₂HPO₄·12H₂O、15.9 mg/mL NaH₂PO₄·2H₂O、10% Trition X-100和0.3 mg/mL NaN₃为金标蛋白保存液;以0.02 mol/L Na₂B₄O₇·10H₂O 含10 mg/mL酪蛋白、5% Trition X-100、0.3 mg/mL NaN₃为样品垫缓冲液;以生理盐水含1.0% Tween-20为样品稀释溶液。制备的FMDV感染与免疫鉴别诊断及免疫评价二联试纸与3ABC阻断ELISA抗体检测试剂盒的符合率为96.20%,与口蹄疫O型抗体液相阻断ELISA检测试剂盒的符合率为94.36%。【结论】通过对试纸生产工艺的优化,建立了稳定的生产工艺,制备的二联试纸检测线显色清晰可见,肉眼识别度高,试纸的检测特异性和敏感性良好。本研究为该试纸的批量生产和产业化应用奠定了基础,为基层口蹄疫的检测提供了稳定、特异、敏感、准确的检测方法。     

大片形吸虫免疫胶体金检测试纸条的研制及初步应用

为建立一种快速、简便、适应现场应用的水牛大片形吸虫检测方法,本试验利用抗大片形吸虫ES抗原单克隆抗体杂交瘤细胞株7D1和7D4分别制备和纯化得到单克隆抗体7D1和7D4;采用柠檬酸三钠还原法制备颗粒大小为30 nm的胶体金,再用胶体金标记纯化单克隆抗体7D1,在硝酸纤维素膜的质控带和检测带处分别包被羊抗鼠IgG二抗和单克隆抗体7D4,经反应条件优化,组装成了大片形吸虫免疫检测试纸条。经测试该检测试纸条对ES抗原的检测下限为0.94 μg/mL,与胰扩盘吸虫、前后盘吸虫、支原体、弓形虫、伊氏锥虫均无交叉反应,特异性好。采集广西地区334份水牛新鲜粪样并用试纸条检测,阳性率为38.62%。结果表明,试纸条方法操作简单、快速、易于判定,特异性好,敏感性高,适合基层大面积普查和现场检测大片形吸虫。     

Preparation and Primary Application of Immunity Colloidal Gold Test Strip for Fasciola gigantica

To establish a rapid, simple, sensitive and adapting field application assay for water buffalo Fasciola gigantica, the monoclonal antibodies of hybridoma cell strains 7D1 and 7D4 against excretory-secretary antigen(ES Ag) of Fasciola gigantica were prepared and purified. Meanwhile, the colloidal gold particle was prepared by the sodium cirrate reduction method, then the affinity purified 7D1 monoclone antibodies was labeled with the 30 nm colloidal gold particles. The 7D4 monoclonal antibodies and the goat anti-mouse IgG antibody using as secondary antibodies were coated on the surface of nitrocellulose filter(NC) membrane as the test line (T line) and control line (C line), respectively. The immune colloidal gold test strip was developed by optimizing the experiment conditions. The test results showed the prepared strip had a detecting prescribed minimum of 0.94 μg/mL of Fasciola gigantica excretory-secretary antigen, and it had no any cross reaction with the antigens of Eurytrema pancreaticum, Paramphistome, Chlamydia, Toxoplasma gondii and Trypanosoma evansi. Using the prepared strip to investigate the 334 fresh feces from buffaloes in Guangxi and the positive rate was 38.62%. These results indicated that this strip method was simple, rapid and easy determination, good specificity, high sensitivity, and adapted field application assay for Fasciola gigantica.     

恩诺沙星完全抗原的合成及兔源多抗血清的制备

为了合成恩诺沙星（enrofloxacin,EFLX）完全抗原,制备兔源多克隆抗体血清,试验通过活化酯法将小分子的EFLX偶联到牛血清白蛋白（BSA）和鸡卵清白蛋白(OVA)上,形成偶联物EFLX-BSA和EFLX-OVA,以EFLX-BSA为免疫原免疫兔,以EFLX-OVA为检测原通过ELISA方法检测兔血清效价。结果表明,免疫兔血清效价都在1∶51200以上,其中2号兔效价最高,达到1∶204800,敏感性好,半数抑制浓度IC₅₀为169.91 ng/mL,检测范围为37.52～302.31 ng/mL。本试验成功制备了EFLX完全抗原,获得了敏感的兔源多克隆抗体血清,为以后ELISA试剂盒和胶体金试纸条的研发奠定了基础。     

A modified live canine parvovirus vaccine. II. Immune response

The safety and efficacy of an attenuated canine parvovirus (A-CPV) vaccine was evaluated in both experimental and in field dogs. After parenteral vaccination, seronegative dogs developed hemagglutination-inhibition (HI) antibody titers as early as postvaccination (PV) day 2. Maximal titers occurred within 1 week. Immunity was associated with the persistence of HI antibody titers (titers greater than 80) that endured at least 2 years. Immune dogs challenged with virulent CPV did not shed virus in their feces. The A-CPV vaccine did not cause illness alone or in combination with living canine distemper (CD) and canine adenovirus type-2 (CAV-2) vaccines, nor did it interfere with the immune response to the other viruses. A high rate (greater than 98%) of immunity was engendered in seronegative pups. In contrast, maternal antibody interfered with the active immune response to the A-CPV. More than 95% of the dogs with HI titers less than 10 responded to the vaccine, but only 50% responded when titers were approximately 20. No animal with a titer greater than 80 at the time of vaccination became actively immunized. Susceptibility to virulent CPV during that period when maternal antibody no longer protects against infection, but still prevents active immunization, is the principal cause of vaccinal failure in breeding kennels where CPV is present. Reduction, but not complete elimination, of CPV disease in large breeding kennels occurred within 1-2 months of instituting an A-CPV vaccination program.     

H9N2亚型禽流感病毒中和单克隆抗体的制备与应用

为建立H9N2亚型禽流感病毒（Avian influenza virus,AIV）免疫层析快速检测技术,本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠,将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养;以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法,通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体;以胶体金标记HA单克隆抗体,配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线,制备H9N2亚型AIV快速检测试纸条,测定其特异性和敏感性。结果显示,获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞,其单克隆抗体腹水IPMA效价在1.28×10^-4至2.56×10^-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性,其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性,中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示,该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2,灵敏度与经典血凝试验（HA）相当,与其他亚型AIV （H1、H3、H5、H7）,以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体,并初步研制了H9N2亚型AIV检测试纸条,为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。     

抗猫泛白细胞减少症病毒单克隆抗体的制备及其生物学特性鉴定

用 PEG60 0 0沉淀和蔗糖密度梯度离心从细胞培养物中纯化猫泛白细胞减少症病毒 ( FPV) ,以纯化FPV免疫 BALB/c小鼠 ,运用淋巴细胞杂交瘤技术 ,获得了 4株抗 FPV的特异性单克隆抗体 ( Mc Ab)。其腹水 Mc Ab的 ELISA效价在 1 0 - 4～ 1 0 - 5之间 ,其中 1株具有血凝抑制能力。经 ELISA阻断试验及 ELISA交叉反应性试验测定 ,这 4株 Mc Ab可与 FPV、犬细小病毒 ( CPV)和水貂肠炎病毒 ( MEV)呈特异性反应 ,因此可作为检测 FPV、CPV和 MEV共同抗原的通用试剂     

Response of pups with maternal derived antibody to modified-live canine parvovirus vaccine

The AA reports the results of vaccination against canine parvovirus (CPV) of pups with maternal antibody, utilizing a modified-live virus (MLV) CPV vaccine having a titer of 10⁷ TCID₅₀/dose. This vaccine was shown to be effective also when HI antibody titers of pups were ≤ 1:80.     

猪血凝性脑脊髓炎病毒不同佐剂灭活疫苗免疫BALB/c小鼠的比较试验

用血凝/血凝抑制试验（HA/HI）检测自制的猪血凝性脑脊髓炎病毒（HEV）氢氧化铝胶灭活苗和蜂胶佐剂灭活苗免疫BALB/c小鼠后的抗体效价,依据小鼠抗体效价比较不同免疫佐剂的效果。结果表明,用氢氧化铝胶佐剂配制的疫苗可刺激接种动物产生快速免疫应答反应,抗体产生效价高、维持时间长,其效果优于其它佐剂,有望成为HEV灭活疫苗的候选佐剂。     

鉴别布鲁菌病自然感染动物与疫苗免疫动物胶体金检测试纸条的研制

用金黄色葡萄球菌A蛋白（SPA）标记胶体金技术制作金标垫,以亲和层析法纯化的BP26蛋白作为检测抗原（T线）,研制胶体金快速检测试纸条。用建立的试纸条检测小鼠布鲁菌感染血清,可成功鉴别牛布鲁菌S19感染和BP26缺失疫苗株YZ-2免疫的小鼠。试纸条检测与布鲁菌属同源性较近的几株细菌的阳性血清,结果无交叉反应;试纸条敏感性高于虎红平板凝集试验检测方法,近似于ELISA方法;准确性同于ELISA法,且更为简便快捷。该试纸条具有鉴别布鲁菌病自然感染和疫苗免疫的应用前景。     

Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections

An enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of CPV-specific mouse monoclonal antibodies, which recognise different epitopes of the haemagglutinin of CPV and which also neutralise the virus. A double antibody sandwich (DAS) ELISA for the detection of CPV in dog faeces was compared with the haemagglutination (HA) test. The DAS-ELISA proved to be more specific, sensitive and easier to perform than the HA assay. An indirect ELISA and a competitive ELISA for the detection of CPV-specific antibodies in dog sera were compared with the haemagglutination inhibition (HI) test. Both ELISA systems proved to be specific and easy-to-use methods for the detection of CPV-specific antibodies. The indirect ELISA, specially, proved to be more sensitive than the HI test. The higher sensitivity and specificity of the ELISA's as compared to HA and HI tests, and their ease of use, make them suitable for routine use in the serology and diagnosis of CPV infections.