CN104459142B - 一种新城疫病毒强毒株和弱毒株鉴别检测试纸 - Google Patents
一种新城疫病毒强毒株和弱毒株鉴别检测试纸 Download PDFInfo
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Abstract
本发明涉及一种家禽疫病感染与免疫的鉴别检测器具,特别是涉及一种新城疫病毒强毒和弱毒鉴别检测试纸,由支撑板,样品垫,金标垫,检测膜和吸水垫构成,检测膜上含有强毒检测线T1“│”,弱毒检测线T2“│”和质控线C“│”印迹。鉴别检测时,在检测膜显现三条红色条带“│││”为新城疫强毒感染,两条红色条带“││”为新城疫弱毒疫苗免疫,只显现一条红色条带“│”为新城疫病毒阴性。本鉴别检测试纸特异性强,敏感性高,反应谱广,可检测现有弱毒疫苗株和和多数流行强毒株,且操作简便、快速,可广泛用于新城疫病毒感染与免疫的鉴别检测,易于在生产实践中推广应用。
Description
技术领域
本发明涉及一种家禽疫病感染与免疫的鉴别检测器具,特别是涉及一种新城疫病毒强毒和弱毒鉴别检测试纸。
背景技术
鸡新城疫(Newcastle disease, ND)又称亚洲鸡瘟,是由新城疫病毒(Newcastledisease virus, NDV)引起的一种以呼吸道、消化道粘膜出血为典型病变的高度接触性、急性败血性禽类传染病。除家禽外,至少有200 多种鸟可以自然或实验室感染,是危害世界养禽业的最严重的传染病之一。该病自1926 年首次报道以来,至今仍在世界各地流行,时有暴发,给养禽业造成了严重的经济损失,世界卫生组织(OIE)将其列为应呈报疫病,我国将其列为一类动物传染病。我国采用弱毒疫苗预防控制了ND的大规模流行,但ND的流行和发病特点又发生新变化,多表现为非典型和慢性感染,出现了所谓“非典型新城疫”和“温和型新城疫”,同时NDV与禽流感病毒等其它呼吸道病原混合感染也十分普遍,使ND 此起彼伏,给养禽业带来了不小的损失,也使新城疫的防制更加困难。NDV为有囊膜、不分节段、单股负链RNA病毒,属于副黏病毒科(Paramyxoviridae)禽副黏病毒属(Paramyxovirus),其血清型为禽副黏病毒I型(APMV-1),又可分为16个基因型。NDV基因组含有15186、15192或15198个核苷酸,病毒基因组结构模式为3’-NP-P-M-F-HN-L-5’,依次编码核衣壳蛋白(Nucleocapsid protein, NP)、磷蛋白(Phosphoprotein, P)、基质蛋白(Matrix protein,M)、融合蛋白(Fusion protein, F)、血凝素-神经氨酸酶蛋白(Heamagglutinin-Neuraminidase protein, HN)和大RNA依赖RNA聚合酶(Large RNA dependent RNA-polymerase, L),其中F和HN蛋白是NDV表面重要的两个囊膜糖蛋白,它们在病毒感染过程中起到重要的作用。NDV病毒毒力主要取决于F前体蛋白部分氨基酸序列,高致病性NDV通常含有C-112R/K-RQ/K/R-R/K-R-F117-N,而低致病性NDV则为C-112G/EK/R-Q-G/E-R-L117-N,世界动物卫生组织将NDV分为嗜内脏型和神经型(高死亡率)、中发型(低死亡率、中度呼吸***症状)、弱毒型(温和呼吸***感染、不死亡,疫苗株)和无症状型(无症状或肠道亚临床感染)。基因I型毒株均为弱毒株,在基因II型病毒中,除弱毒株外还有中等毒力毒株和高致病力的强毒株,而在基因I和II型毒株之后出现的其它7个基因型(III-IX型)NDV 均为强毒株。流行病学数据表明,上世纪80年代在我国鸡群中主要流行的NDV 毒株为VIf和VIg亚型,90 年代中期我国出现了NDV 基因VII型,并且成为我国NDV流行的优势基因型,当前VIId亚型在我国流行强毒株中已占有绝对优势,而我国禽群中普遍使用的弱毒疫苗株LaSota属于基因II型,因此新城疫疫苗株与当前流行毒株之间的基因型和抗原性差异是鉴别新城疫病毒疫苗株和强毒株的理论依据。
目前,我国新城疫的防控仍然采取以疫苗免疫为主的策略,上世纪90年代以来ND弱毒疫苗株在我国得到了大规模、高剂量的频繁使用,常用疫苗株LaSota对不同基因型的ND 强毒流行株虽能产生完全的临床保护,便不能防止流行株在免疫鸡中的感染复制和排出,而免疫鸡群中ND 强毒感染仍然时有发生,同时由于缺乏血清学标志和配套的鉴别诊断方法,其在我国的大规模应用使得通过血凝抑制试验(HI)和酶联免疫吸附试验(ELISA)等抗体检测技术很难区分免疫弱毒和野毒感染,不利于ND的控制和净化。随着不同来源、不同地区NDV野毒株和疫苗毒株全基因序列的完成和分子生物学检测技术的发展,国内外学者在NDV鉴别检测方面进行了大量研究,通过分析NDV基因组的强、弱毒株特征性位点,分别设计强、弱毒株特异性引物或探针,建立了区分NDV强毒和弱毒的RT-PCR和荧光定量PCR 等分子鉴别检测方法,具有较高的敏感性和特异性,但这些检测方法需要依赖于PCR仪或荧光定量PCR仪,存在操作相对繁琐、耗时较长、检测成本高等不足,限制了其在流行病学调查和兽医临床中的应用。免疫层析试纸是在单克隆抗体技术、胶体金免疫层析技术和新材料技术基础上发展起来的一种新型体外检测技术,是理想的即时检测(point-of-care test,POCT)和现场检测技术,其具有更加灵敏、特异、简便、快速等优点,尤其是可实现“傻瓜式”操作,即不需要任何附加仪器设备即可进行现场检测,并在1-5分钟内判定结果,广泛应用于各种分析物的定性和半定量快速检测,包括抗原、半抗原、抗体和核酸,已成为当今最快速敏感的免疫学检测技术之一。河南省动物免疫学重点实验室从1995年开始***开展免疫试纸快速检测技术研究,率先在国际上研制成功动物疫病病原和抗体检测胶体试纸产品――鸡传染性法氏囊病快速诊断试纸和猪旋毛虫抗体检测纸,建立了动物疫病快速检测试纸研发技术平台,截至目前已成功开发出动物疫病抗原、抗体以及药物残留检测等系列快速检测试纸产品,大大推动了该技术的应用和发展。本发明针对NDV野毒感染与疫苗免疫鉴别诊断的关键技术问题,筛选鉴定可区分NDV强、弱毒株的高亲和力配对单克隆抗体,建立基于免疫层析试纸的蛋白芯片技术平台,研制新城疫病毒强毒株和弱毒株鉴别检测试纸,建立适用于养殖基层和临床检测的快捷、简便的新城疫野毒感染和疫苗免疫联检技术,实现对禽类新城疫病毒野毒感染的实时监测,为我国新城疫防控与净化提供技术支撑,对新城疫疫情监测和防控有重要意义。
发明内容
本发明的目的是:研制适用于家禽新城疫感染与免疫鉴别检测的新城疫病毒强毒和弱毒鉴别检测试纸,本试纸特异、敏感、快捷、简便,易在生产实践中推广应用。
本发明的技术方案是:一种新城疫病毒强毒和弱毒鉴别检测试纸,由支撑板,样品垫,金标垫,检测膜和吸水垫构成,金标垫为吸附胶体金标记抗新城疫病毒单克隆抗体mAb1的玻璃纤维,检测膜为印迹强毒检测线T1、弱毒检测线T2和质控线C的硝酸纤维素膜,由样品端至手柄端的排列为强毒检测线T1/弱毒检测线T2/质控线C:“│││”,强毒检测线T1为抗新城疫病毒单克隆抗体mAb2印迹“│”,弱毒检测线T2为抗新城疫病毒多克隆抗体pAb1或抗新城疫病毒单克隆抗体mAb3印迹“│”,质控线C为抗小鼠IgG抗体pAb2或金黄色葡萄球菌SPA印迹“│”。鉴别检测时,新城疫强毒感染在检测膜显现强毒检测线T1,弱毒检测线T2和质控线C三条红色条带“│││”,新城疫弱毒疫苗免疫在检测膜显现弱毒检测线T2和质控线C两条红色条带“││”,无新城疫病毒则只显现质控线C一条红色条带“│”。
以新城疫病毒标准强毒F48E8株为免疫抗原,通过杂交瘤细胞技术生产鉴定抗新城疫病毒单克隆抗体,利用免疫过氧化物酶单层细胞试验(IPMA)筛选区分新城疫病毒强毒和弱毒的单克隆抗体,以叠加酶联免疫吸附试验(ELISA)筛选识别不同抗原表位的单克隆抗体。用于胶体金标记的单克隆抗体mAb1和弱毒检测线T2的单克隆抗体mAb3均特异识别新城疫病毒强毒株和弱毒疫苗株,分别识别新城疫病毒的不同抗原表位,用于强毒检测线T2的单克隆抗体mAb2特异识别新城疫病毒强毒株,但不与弱毒株反应。同时,以IPMA筛选与新城疫病毒反应谱广的单克隆抗体,胶体金标记单克隆抗体mAb1和弱毒检测线T2单克隆抗体mAb3可识别新城疫病毒中等毒力和弱毒疫苗以及多数流行强毒株,强毒检测线T1单克隆抗体mAb2可识别多数新城疫病毒流行强毒株。以新城疫病毒弱毒疫苗LaSota 株为免疫抗原制备鸡抗新城疫病毒多克隆抗体pAb1,可用于弱毒检测线T2印迹。以小鼠IgG为免疫抗原制备羊抗小鼠IgG多克隆抗体pAb2,pAb2和金黄色葡萄球菌SPA均可用于质控线C印迹。
本发明有益的积极效果,新城疫病毒强毒和弱毒鉴别检测试纸实现了新城疫病毒强毒和弱毒的同步联检,可有效鉴别家禽新城疫强毒感染与疫苗免疫,并且操作简单,人人都可操作,能较好满足不同层次人员的需要,如疫病监测、海关检疫、卫生防疫、集约化养殖到个体养殖等,易于大范围推广应用,具有广阔的市场前景和较大的经济、社会效益。检测试纸条具有下列各项优点:
(1)强毒和弱毒联检。新城疫病毒强毒和弱毒鉴别检测试纸在检测膜上含有识别新城疫病毒强毒和弱毒的弱毒检测线和识别强毒但不识别弱毒的强毒检测线,可同步进行新城疫病毒的强毒株和弱毒株联检,实现新城疫的强毒和弱毒鉴别检测。
(2)特异性强,敏感性高。新城疫病毒强毒和弱毒鉴别检测试纸以可区分新城疫病毒强毒和弱毒的高亲和力配对单克隆抗体为基础制备而成,单克隆抗体的特异性强和敏感性高,金标抗体中金颗粒与抗体分子之间无共价键形成,二者通过异性电荷间的范德华力相结合,胶体金对标记抗体的反应性影响很小,且具有较高的标记率。因此,鉴别检测试纸具有较高的特异性和敏感性。
(2)操作简便快速。使用鉴别检测试纸时无需任何其它试剂,只要将其***待检样品10秒左右,在2分钟内即可判定检测结果。
(3)显示检测结果形象、直观准确。鉴别检测试纸以显示红棕色“│”、“││”和“│││”印迹作为检测的阴性、弱毒和强毒阳性标记,即在检测膜上显示一条棕红色条带“│”为新城疫病毒阴性,表示被检测样品无弱毒疫苗或强毒感染,两条棕红色条带“││”为新城疫病毒弱毒阳性,表示被检样品为弱毒疫苗免疫,三条棕红色条带“│││”为新城疫病毒强毒阳性,表示被检样品为强毒感染,结果判定形象、直观、准确,简单明了,不易出现假阴性和假阳性误判。
(4)成本低,投资少。使用鉴别检测试纸,不需另配仪器设备及其它试剂,使现场检测一步到位,成本低廉,投资少,见效快。
附图说明 下面结合附图和实施例对本发明进一步说明。
图1是新城疫病毒强毒和弱毒鉴别检测试纸侧视结构示意图。
图2是新城疫病毒强毒和弱毒鉴别检测试纸俯视结构示意图。
图中,1. 支撑板, 2. 样品垫,3. 金标垫,4. 检测膜, 5. 吸水垫,6. 强毒检测线T1印迹,7. 弱毒检测线T2印迹,8. 质控线C印迹,9-1.样品端保护膜,9-2. 手柄端保护膜,10. 标记线。
具体实施方式 新城疫病毒强毒和弱毒鉴别检测试纸可广泛应用于多种家禽(如鸡、鸭、鹅、鸽等)新城疫病毒强毒感染和疫苗免疫的鉴别检测。制备新城疫病毒强毒和弱毒鉴别检测试纸,首先需制备新城疫病毒免疫抗原,进而制备抗新城疫病毒多克隆抗体和单克隆抗体,并筛选区分新城疫强毒和弱毒的单克隆抗体,识别新城疫强毒和弱毒的单克隆抗体mAb1用于制备胶体金标记物,识别新城疫强毒而不识别弱毒的单克隆抗体mAb2用于印制强毒检测线印迹“│”,识别新城疫强毒和弱毒的多克隆抗体pAb1或单克隆抗体mAb3用于印制弱毒检测线印迹“│”,其次需制备羊抗小鼠IgG抗体或金黄色葡萄球菌SPA,用于印制质控线印迹“|”。
(1)新城疫病毒免疫抗原的制备。
以新城疫病毒标准强毒株F48E8经尿囊腔接种9-11日龄SPF鸡胚,36-48小时收取尿囊液,差速离心纯化病毒抗原,4℃ 3000 r/min低速离心30 min去除杂质,4℃ 60000 r/min超速离心1 h,用7 ml 0.01 mol/L PBS (pH值7.2)重悬沉淀。以血凝试验(HA)测定新城疫病毒的HA效价达2-12以上。
(2)抗新城疫病毒单克隆抗体的制备。
(2.1)杂交瘤细胞株的建立。
将新城疫病毒免疫抗原与弗氏免疫佐剂等量混合,充分乳化,以50mg-100mg/只免疫BALB/c系小鼠3次,每次间隔15-30天;第3次加强免疫后3-4天,将免疫小鼠眼球放血,拉颈致死,于75%酒精浸泡5-10min,无菌取其脾细胞;剪碎并经100目尼龙网过滤,1000 r/min离心10 min,收集脾细胞;将1×108的脾细胞与2-5×107的SP2/0骨髓瘤细胞混合,1000 r/min离心10 min,弃上清,在37℃的水浴中将0.7-1 ml的40%-50% PEG 4000(pH8.5-9.0)缓缓加入细胞,温育1 min后,缓慢加入无血清1640培养基15 ml,以终止PEG的作用,37℃水浴5-10 min,1000 r/min离心10 min,弃上清,将细胞重悬于HAT选择培养基中,并加入96孔培养板(100 ml-200 ml/孔),置37℃ 5% CO2培养箱中培养。培养7-10天后,取杂交瘤细胞培养上清以免疫过氧化物酶单层细胞试验(IPMA)筛选阳性杂交瘤细胞。以新城疫病毒标准强毒F48E8株感染鸡胚成纤维细胞(CEF)或仓鼠肾细胞(BHK-21)细胞,经甲醇固定后,5%脱脂奶37℃封闭1 h;加待检细胞培养上清50mL/孔,设HAT培养基和小鼠免疫血清为阴性和阳性对照;加1:500 辣根过氧化物酶(HRP)标记羊抗小鼠IgG抗体(50 mL/孔),37℃作用30 min;每步反应后均用含0.05% Tween-20的PBS充分洗涤;以底物AEC室温显色10-20 min,用水冲洗中止显色后,在显微镜下观察显色结果。选取强阳性、细胞生长旺盛的克隆孔进行连续3次有限稀释克隆化,扩大培养后冻存细胞,建立抗新城疫病毒单克隆抗体杂交瘤细胞株28株。
(2.2)单克隆抗体的制备:以体内诱生腹水制备单抗。取经降殖烷或液体石蜡致敏的经产Balb/c小鼠,腹腔注射对数生长期的杂交瘤细胞107个/只,7 d~10 d后抽取腹水,离心后取上清,分装,冻存。
(2.3)单克隆抗体的鉴定
(2.3.1)单克隆抗体的抗体效价
以免疫过氧化物酶单层细胞试验(IPMA)测定杂交瘤细胞培养上清和腹水的单抗效价,单抗上清和腹水的IPMA效价分别在1:16和1:1600以上。以血凝抑制试验(HI)测定单克隆抗体腹水的HI效价,筛选具有血凝抑制活性单克隆抗体5株,其HI效价在1:128以上。
(2.3.2)单克隆抗体的特异性
用NDV标准毒株、流行毒株和弱毒疫苗株I系(Mukteswar株)、II系(B1株)、III系(F株)、IV系(Lasota株)、V4(耐高温株)和克隆株(Clone-30、N-29、Clone-83和N-88)感染鸡胚成纤维细胞(CEF)或仓鼠肾细胞(BHK-21),以免疫过氧化物酶单层细胞试验(IPMA)测定单抗与NDV流行毒株的反应性,筛选获得同时特异识别NDV标准毒株、流行毒株和弱毒疫苗株的单克隆抗体22株,而特异识别NDV标准毒株和流行毒株但不识别弱毒疫苗株的单克隆抗体6株。以免疫过氧化物酶单层细胞试验(IPMA)检测上述28株单克隆抗体与疫苗株和流行毒株的反应谱,证明用于胶体金标记和弱毒检测线T2的单克隆抗体可识别新城疫病毒中等毒力和弱毒疫苗以及多数流行强毒株,用于强毒检测线的单克隆抗体可识别多数新城疫病毒流行强毒株,均具有较广的NDV反应谱。
(2.3.3)单克隆抗体识别抗原表位分析
以阻断ELISA或叠加ELISA对NDV单克隆抗体识别的抗原表位进行分析,筛选获得分别识别新城疫病毒不同抗原表位的单克隆抗体mAb1和mAb3,其分别用于胶体金标记和弱毒检测线T2印迹;获得特异识别新城疫病毒标准毒株和流行毒株,而不与弱毒疫苗株反应的单克隆抗体mAb2,用于强毒检测线T1印迹。
(2.3.3.1)阻断ELISA:以HRP分别标记单克隆抗体,ELISA阻断试验鉴定单克隆抗体识别抗原表位的特异性,首先在NDV标准阳性抗原包被的酶标板中逐行加入未标记的单抗,设NDV阳性血清和PBS对照,37℃作用1 h;然后逐列加入HRP标记的单抗,37℃作用1 h;以底物TMB进行显色,读取各检测孔的OD450值。显著抑制酶标单抗结合的未标记单抗识别相同或相近的抗原表位,而对酶标单抗结合无明显影响的未标记单抗则识别不同的抗原表位。
(2.3.3.2) 叠加ELISA:首先利用包被抗原的酶标板以间接ELISA测定各单抗的工作浓度,绘制抗原饱和曲线。在叠加ELISA试验中,根据抗原饱和曲线适当稀释单抗,并配对加入酶标板各孔,37℃孵育1 h~2 h;分别加入酶标二抗和TMB进行显色,读取各孔OD450值,按下列公式计算叠加系数(AI):AI=[2´OD1+2/(OD1+OD2)-1] ´100%,其中OD1+2为配对单抗孔的OD450值,OD1和OD2为两个独立单抗孔的OD450值。两个单抗的AI值小于40%判为识别相同或相近抗原表位;AI值大于40%判为识别不同抗原表位。
(2.4)单克隆抗体的纯化:以辛酸-硫酸铵法从小鼠腹水中纯化单抗IgG。取1 mL小鼠腹水,加入2 mL 0.06 mol/L乙酸钠缓冲液(pH 5.0),以0.1 mol/L HCl调至pH 4.5;于室温搅拌下,逐滴加入33 mL辛酸,4℃静置2 h,15000 r/min离心30 min,弃沉淀;在离心上清中加入1/10体积0.01 mol/L PBS (pH7.4),以0.1 mol/L NaOH调至pH 7.4;于冰浴条件下加入饱和硫酸铵至终浓度45%,4℃静置2 h,10000 r/min离心30 min,弃上清;以适量PBS重悬沉淀,对PBS透析过夜,换液3次。以分光光度计法或考马斯亮蓝染色法(Bradford法)测定纯化单克隆抗体IgG的蛋白含量在1mg/ml以上,以免疫过氧化物酶单层细胞试验(IPMA)测定小鼠腹水和纯化IgG的抗体效价在1:1000以上,分装,冻存。
(3)抗新城疫病毒多克隆抗体的制备。
以50mg~100mg/kg体重的新城疫病毒免疫抗原加免疫佐剂经皮下和肌肉注射免疫SPF鸡3~4次,末次免疫10天后,静脉采血,以IPMA测定其血清抗体效价在1:1000以上时,心脏采血或颈动脉放血,收集高免血清,以无水硫酸钠(Na2SO4)提取免疫家禽血清IgY,取1份免疫血清加2份0.01 mol/L PBS(pH7.2)混合,加入无水Na2SO4至终浓度18%,37℃水浴30min,4000 r/min离心20min,弃上清,以适量PBS(pH7.2)重悬沉淀,加终浓度16%的Na2SO4,37℃沉淀30min,4000r/min离心20min,弃上清,再以适量PBS(pH7.2)重悬沉淀,以终浓度14%的Na2SO4沉淀,4000r/min离心20min,弃上清,以适量PBS(pH7.2)重悬沉淀,对PBS(pH7.2)过夜透析,换液2~3次,测定抗体效价和蛋白浓度(10-20 mg/ml),所制备抗新城疫病毒多克隆抗体pAb1可用于检测试纸弱毒检测线T2印迹的印制。
(4)兔抗小鼠IgG多克隆抗体的制备
以纯化小鼠IgG免疫2.0 kg左右健康新西兰兔,首次免疫以弗氏完全佐剂乳化抗原,皮下多点注射50 mg/只,每次加强免疫间隔3 w,以弗氏不完全佐剂乳化抗原肌肉注射,最后一次加强免疫2 w后,以琼脂扩散试验(AGP)测定免疫血清抗体效价高于1:40时,采集高免兔全血,分离血清,以辛酸-硫酸铵法纯化兔抗小鼠IgG,方法同(2.4)单抗纯化,辛酸用量为45 mL/mL血清,测定抗体效价和蛋白浓度(10-20 mg/ml),所制备兔抗小鼠IgG多克隆抗体pAb2可用于检测试纸质控线C印迹的印制。
(5)单克隆抗体的胶体金标记
(5.1)胶体金的制备:取100 mL超纯水置于500 mL洁净的锥形瓶,加入1 mL 1 %(w/v)氯金酸煮沸;在搅拌状态下迅速加入新鲜配制的1 mL 1%(w/v)柠檬酸钠溶液,煮沸约3 min至溶液颜色由黄色变为***,继续煮沸2 min;待溶液凉至室温,补超纯水至100mL,以0.2 mol/L K2CO3调pH至9.0,4℃避光可保存数月。
(5.2)最适标记蛋白浓度测定:取待标记抗NDV单抗IgG对20 mmol/L硼酸钠溶液(pH 8.0)4℃过夜透析。在微孔板中以25 mL超纯水 1:2、1:4、1:8……倍比稀释待标记NDV单抗;各孔加入125 mL胶体金溶液,RT静置5 min;加入125 mL 1 mol/L NaCl溶液;各孔颜色随蛋白浓度的降低而由红色变为蓝色。以颜色未变蓝的单抗最高稀释度的蛋白浓度为胶体金最适标记浓度,胶体金标记时,蛋白浓度增加20%。
(5.3)单克隆抗体的胶体金标记:取2 mL 最适蛋白浓度的待标记单抗IgG,加入10mL胶体金溶液(pH 9.0),迅速混匀,室温作用10 min~15 min;加入1/10体积含10% (w/v)牛血清白蛋白(BSA)的20 mmol/L硼酸钠溶液,迅速混匀,RT作用10 min~15 min;4℃15000 g离心30min,小心移去上清;以含1% (w/v)BSA 的20 mmol/L硼酸钠溶液重悬胶体金,同上离心,弃上清;重复洗涤1次,以1 mL含1% (w/v)BSA的20mmol/L硼酸钠溶液重悬胶体金,4℃保存备用。
(6)检测膜的制备
将硝酸纤维素检测膜切成2.5×30 cm2的长条,置于XYZ 3000喷点仪平台上,并以压条固定;用PBS(pH 7.2)将特异识别新城疫病毒强毒株但不与弱毒株反应的单抗、特异识别新城疫病毒强毒株和弱毒疫苗株的单抗或多抗IgG和兔抗鼠IgG稀释至 1 mg/mL,并分别放于贮存池;利用Biojet Quanti 3000以1 mL/cm分别将抗NDV单抗或多抗IgG溶液和兔抗鼠IgG溶液喷点于检测膜中央,形成强毒检测线T1、弱毒检测线T2和质控线C印迹,检测线与质控线相距0.5 cm;置42℃干燥箱30 min或室温自然干燥;将检测膜置塑料袋,加干燥剂4℃密闭保存备用。
(7)结合垫的制备
将玻璃棉切成1.5×30 cm2的长条,置于XYZ 3000喷点仪平台上,并以压条固定;取1 mL胶体金标记物加入2 mL含2% (w/v) BSA、3% (w/v)蔗糖、0.6 mol/L NaCl、0.2%Tween 20 (v/v)和 0.1% (w/v)叠氮钠的20 mmol/L硼酸钠溶液(pH 8.0);利用AirjetQuanti 3000以15 mL/cm将抗胶体金标记物溶液喷点于玻璃棉;置50℃干燥箱30 min干燥;将胶金垫置塑料袋,加干燥剂4℃密闭保存备用。
(8)样品垫的制备
将玻璃棉切成1.5×30 cm2的长条,以将含0.1 mol/L NaCl、0.2% Tween 20 (v/v)和 0.1% (w/v)叠氮钠的PBS (pH 7.2)溶液浸泡玻璃棉条;置50℃干燥箱30 min干燥;将样品垫置塑料袋,加干燥剂RT密闭保存备用。
(9)吸水垫的制备
将玻璃棉切成2.5×30 cm2的长条,将吸水垫置塑料袋,加干燥剂RT密闭保存备用。
(10)支撑板的制备
将双面胶贴于PVC支撑板,切成7.5×30 cm2的长板,制备支撑板。
(11)试纸的组装
利用LM5000试纸装配仪或手工将上述材料装配成试纸板。先将检测膜粘贴于支撑板中央,然后将胶金垫和样品垫依次粘贴于检测膜的样品端,各层间重叠1 mm~2 mm,再将吸水垫粘贴于检测膜的另一端,与检测膜重叠1 mm~2 mm,在样品端以白色塑胶膜包裹样品垫和胶金垫,塑胶膜上印有检测方向及检测溶液上限标记,在手柄端以蓝色塑胶膜包裹吸水垫。
(12)切割与包装
利用CM4000切割仪将装配好的试纸板切成0.3 cm的试纸条,将试纸条分装入塑料袋,加干燥剂4℃密闭保存。
(13)新城疫病毒强毒株和弱毒株鉴别检测试纸的实施结构
参见图1,图2,图中,1为支撑板用不吸水薄片条,实施中可采用塑胶薄片条或采用不吸水的硬质纸片材,反应试剂载体吸附层由2,3,4,5,组合而成,从样品端2开始,到手柄端5依次粘贴在支撑层 1上面;其中 2为样品端样品垫,实施中可使用玻璃纤维棉简称玻璃棉,3为吸附胶体金标记识别新城疫病毒强毒和弱毒株单克隆抗体mAb1的金标玻璃纤维棉,可采用精制玻璃纤维棉,简称为金标垫,4为检测膜,实施中可采用硝酸纤维素膜,5为手柄端吸水垫,采用吸水纸,如滤纸或其它吸水纸均可,试纸条总长8 cm,宽度0. 4 cm,6为用特异识别新城疫病毒强毒株但不与弱毒株反应的单克隆抗体mAb2在硝酸纤维素膜上印制的强毒检测线印迹“|”,7为特异识别新城疫病毒强毒株和弱毒疫苗株的单克隆抗体mAb3或抗新城疫病毒多克隆抗体pAb1在硝酸纤维素膜上印制的弱毒检测线印迹“|”,8为兔抗小鼠IgG多克隆抗体pAb2或金黄色葡萄球菌SPA在硝酸纤维素膜上印制的质控线印迹“|”,在检测膜上的强毒检测线印迹、弱毒检测线印迹和质控线印迹组合排列为“|||”。9为保护膜,覆盖在玻璃棉及金标玻璃棉的保护膜为9-1,覆盖在吸水层滤纸上的保护膜为9-2。在玻璃棉和金标玻璃棉交界处对应位置的白色保护膜上偏向玻璃棉一方0.5 cm处的保护膜上印有一条标记线10,在标记线10右边印有箭头和Max字样,手柄端保护膜可用黄色或其它颜色,2,3,4,5各层彼此之间交界处纤维互相交叉渗透。
(14)新城疫病毒强毒株和弱毒株鉴别检测试纸实施检测反应原理
当新城疫病毒强毒株和弱毒株鉴别检测试纸样品端***待检测样品溶液后,待检溶液通过虹吸带动待检NDV抗原及金标抗体mAb1一起向硝酸纤维素膜扩散,并最终渗透到滤纸层中,在扩散过程中金标抗体mAb1与待检NDV强毒株或弱毒株抗原相结合,形成金标抗体-抗原复合物,NDV强毒株的金标复合物可同时与检测膜上的强毒检测印迹mAb2和弱毒检测印迹mAb3结合,生成红棕色“||”标记,部分未与抗原结合的金标抗体不能与检测印迹结合而继续扩散,在检测膜上与质控印迹中的pAb2或SPA,生成红棕色标记“|”,两种标记组合叠加,形成三条红棕色阳性标记“|||”,表示样品中含有新城疫强毒;而NDV疫苗毒株的金标复合物不能与检测膜上的强毒检测印迹mAb2结合,只能与弱毒检测印迹mAb3结合,生成红棕色“|”标记,部分未与抗原结合的金标抗体不能与检测印迹结合而继续扩散,在检测膜上与质控印迹中的pAb2或PSA,生成红棕色标记“|”,两种标记组合叠加,形成两条红棕色阳性标记“||”,表示样品中只含有新城疫疫苗毒;当样品中不含新城疫病毒抗原时,没有金标抗体-抗原复合物形成,不能与强毒检测印迹和弱毒检测印迹结合,则生成阴性标记“|”。如果检测膜上没有红棕色标记显示,则表明试纸条已失效。
(15)新城疫病毒强毒株和弱毒株鉴别检测试纸检测实例操作方法
(15.1)检测样品溶液的制备:采集待检活禽拭子(咽拭和肛拭)、病(死)禽组织、粪便和疫苗等不同检测样本,加适量PBS或水进行简单混悬或研磨
(15.2)试纸检测:将新城疫病毒强毒株和弱毒株鉴别检测试纸样品端浸入待检溶液10 s~20 s;取出试纸水平放置5 min~10 min观察结果。
(15.3)检测结果判定:试纸显现三条红棕色条带(强毒检测线、弱毒检测线和质控线)“|||”为NDV强毒阳性,表示待检样品中含有NDV强毒抗原;两条红棕色条带(弱毒检测线和质控线)“||”为NDV弱毒阳性,表示待检样品中含有NDV疫苗毒抗原;仅显现一条红棕色条带(质控线)“|”为NDV阴性,表示待检样品未检出NDV抗原;试纸未显现任何条带表明检测操作不当或试纸失效,需以另取检测试纸重新检测。
实施例一,家禽新城疫强毒感染快速诊断,采集病(死)禽的病变组织,包括腺胃、肺、肝、脾、肾、气管、小肠、大肠和***等,以1:5或1:10加适量PBS或水进行简单研磨,制备待检溶液,以新城疫病毒强毒株和弱毒株鉴别检测试纸按(15)操作方法进行检测和结果判定,鉴别诊断病(死)禽强毒感染或疫苗免疫。试纸显现三条红棕色条带(强毒检测线、弱毒检测线和质控线)“|||”为NDV强毒阳性,表示待检家禽为NDV强毒感染;两条红棕色条带(弱毒检测线和质控线)“||”为NDV弱毒阳性,表示待检家禽为NDV疫苗免疫;仅显现一条红棕色条带(质控线)“|”为NDV阴性,表示待检家禽无强毒感染或疫苗免疫;试纸未显现任何条带表明检测操作不当或试纸失效,需以另取检测试纸重新检测。
实施例二,活禽的新城疫病毒检验检疫,采集活禽拭子(咽拭和肛拭)或粪便,以1:5或1:10加适量PBS或水进行简单混悬,制备待检溶液,以新城疫病毒强毒株和弱毒株鉴别检测试纸按(15)操作方法进行检测和结果判定,鉴别检测活禽的强毒污染或疫苗免疫情况。试纸显现三条红棕色条带(强毒检测线、弱毒检测线和质控线)“|||”为NDV强毒阳性,表示待检家禽或环境为NDV强毒污染;两条红棕色条带(弱毒检测线和质控线)“||”为NDV弱毒阳性,表示待检家禽或环境无NDV强毒污染,为NDV疫苗免疫;仅显现一条红棕色条带(质控线)“|”为NDV阴性,表示待检家禽无强毒感染或疫苗免疫;试纸未显现任何条带表明检测操作不当或试纸失效,需以另取检测试纸重新检测。
实施例三,新城疫弱毒疫苗的质量检测,以1:5或1:10加适量PBS或水溶解新城疫弱毒疫苗,包括新城疫病毒中等毒力I系(Mukteswar株)和弱毒疫苗株II系(B1株)、III系(F株)、IV系(Lasota株)、V4(耐高温株)和克隆株(Clone-30、N-29、Clone-83和N-88)等,制备待检疫苗溶液,以新城疫病毒强毒株和弱毒株鉴别检测试纸按(15)操作方法进行检测和结果判定,检测新城疫弱毒疫苗的病毒含量和鉴别检测疫苗的强毒污染情况。试纸显现三条红棕色条带(强毒检测线、弱毒检测线和质控线)“|||”为NDV强毒阳性,表示待检疫苗为NDV强毒污染;两条红棕色条带(弱毒检测线和质控线)“||”为NDV弱毒阳性,表示待检疫苗无NDV强毒污染,弱毒检测线的显色强度与NDV疫苗含量成正比;仅显现一条红棕色条带(质控线)“|”为NDV阴性,表示待检疫苗无NDV强毒污染,同时也不含疫苗毒;试纸未显现任何条带表明检测操作不当或试纸失效,需以另取检测试纸重新检测。
Claims (3)
1.一种新城疫病毒强毒和弱毒鉴别检测试纸,由支撑板,样品垫,金标垫,检测膜和吸水垫构成,其特征是:金标垫吸附胶体金标记抗新城疫病毒单克隆抗体mAb1,检测膜的强毒检测线T1为抗新城疫病毒单克隆抗体mAb2印迹“│”,弱毒检测线T2为抗新城疫病毒多克隆抗体pAb1或单克隆抗体mAb3印迹“│”,质控线C为抗小鼠IgG抗体pAb2或金黄色葡萄球菌SPA印迹“│”;所述单克隆抗体mAb1和mAb3均特异识别新城疫病毒强毒株和弱毒疫苗株,分别识别新城疫病毒的不同抗原表位;所述单克隆抗体mAb2特异识别新城疫病毒强毒株,且不与弱毒株反应;所述单克隆抗体mAb1、mAb2和mAb3是以新城疫病毒标准强毒F48E8 株为免疫抗原,通过建立杂交瘤细胞的方法制备鉴定出抗新城疫病毒单克隆抗体,利用免疫过氧化物酶单层细胞试验筛选区分出新城疫病毒强毒和弱毒的单克隆抗体,以叠加酶联免疫吸附试验筛选识别出不同抗原表位的单克隆抗体。
2.根据权利要求1所述的鉴别检测试纸,其特征是:新城疫强毒感染时在检测膜显现强毒检测线T1,弱毒检测线T2和质控线C三条红色条带“│││”,新城疫弱毒疫苗免疫时在检测膜显现弱毒检测线T2和质控线C两条红色条带“││”,无新城疫病毒则只显现质控线C一条红色条带“│”。
3.根据要求1或2所述的鉴别检测试纸,其特征是:鉴别检测试纸的新城疫病毒反应谱广,弱毒检测线T2可检测新城疫病毒中等毒力I系和弱毒疫苗株II系、III系、IV系以及多数流行强毒株,强毒检测线T1可检测多数新城疫病毒流行强毒株,可实现对新城疫病毒主要流行毒株与疫苗株的鉴别检测。
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