CN103941003B - 猪李氏杆菌、猪坏死杆菌及猪土拉杆菌三联检测试纸条 - Google Patents
猪李氏杆菌、猪坏死杆菌及猪土拉杆菌三联检测试纸条 Download PDFInfo
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Abstract
本发明涉及一种猪三种细菌病的检测器具,特别是涉及一种猪的李氏杆菌病、坏死杆菌病、土拉杆菌病快速诊断试纸条,试纸条含支撑层、反应试剂载体吸附层,支撑层为不吸水薄片条,反应试剂载体吸附层粘贴于支撑层上,从样品测试端依次为纤维层,上述三种细菌病抗原金标单抗或多抗纤维层,纤维素膜层,手柄端为吸水材料层;分别用上述三种细菌病抗原配对单抗或多抗或单抗溶液在纤维层上喷检测印迹“<b>|||</b>”、“<b>/ / /</b>”、或“<b>\\\</b>”,分别用羊(兔)抗小鼠或猪IgG的多抗或用SPA溶液在纤维素膜层上喷对照印迹“<b>|</b>”、“<b>/</b>”或“<b>\</b>”。该试纸条特异、敏感、直观、准确、简便、快速,能在畜禽饲养、肉类加工和检疫等相关部门推广应用。
Description
一、技术领域
本发明是一种涉及猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病检测试剂显示器具,特别是涉及一种可同时检测猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病的三联检测试纸条。
二、技术背景
李氏杆菌病是由李氏杆菌引起的畜、禽、鼠及人共患的传染病。病原菌对环境条件的抵抗力很强,但常见消毒剂都能很快将其杀死。本病多发于哺乳仔猪及断奶不久的保育猪,通常呈散发,发病率低,病死率高为其特点。本病具有广泛的易感动物群体,因此,传染源也较多,其中带菌的鼠类在疾病传播中常起重要作用。症状可分为败血型、脑膜脑炎型,但常见的是二者混合型。多数病猪兴奋不安,运动失调,肌肉震颤、无目的地运动。有的病猪头颈后仰,四肢张开呈观星姿势。有的后肢麻痹,拖地不能站立。有的阵发性痉挛,侧卧、四肢乱划,呈游泳状。病初体温升高41~42℃,食欲减少。粪干尿少,后期降致常温或常温以下。剖检:有神经症状病猪,脑及脑膜充血、水肿,脑脊髓液增加,稍混浊,内含较多细胞。脑干变软有小脓灶。组织学检查,见严重的单核细胞浸润现象。败血症严重病猪,肺充血、水肿,气管、支气管常有泡沫样液体。肝有灰白色小坏死灶,心肌柔软,内外膜有出血点,肠系膜淋巴结肿大。
猪坏死杆菌病是由坏死杆菌引起猪的一种慢性传染病。本病的主要传染源是病畜,禽类和哺乳动物都可感染,感染途径是损伤的皮肤和粘膜。在多雨、潮湿、拥挤和卫生条件低劣的情况下容易发生。潜伏期为1~3天,长的可达2周,主要发生于仔猪和架子猪,一般呈散发性或地方性流行,猪以坏死性皮炎多见,主要表现在颈部、胸侧、臀部皮下脂肪较多的皮肤间或在耳根和四肢下部的皮肤发生小而突起的丘疹。上面覆盖有容易剥离的痂皮,痂皮下组织迅速坏死溃烂,皮下形成很大的囊状溃烂区,流出大量的灰黄色液体,并有特殊臭味。随后皮肤发生溃烂,严重者病变深入肌层,甚至穿透腹壁,形成瘘管。其次是坏死性口炎,常见于仔猪,表现在唇、齿龈及颚部粘膜发生溃疡,上面覆盖坏死组织。坏死性肠炎,病猪常与仔猪副伤寒并发。表现大肠、小肠粘膜坏死,形成白色假膜,假膜下为不规则的溃疡,导致病猪生前有严重腹泻,迅速消瘦等全身症状。此外有的还表现坏死性鼻炎,常继发于萎缩性鼻炎,主要见于仔猪和幼猪。
土拉杆菌病是土拉费朗西斯菌引起的一种人兽共患急性传染病。猪感染土拉杆菌病的主要特征是患猪体温升高、淋巴结肿大、支气管肺炎,发病率和病死率均不高。土拉费朗西斯菌为革兰氏阴性多形态的细菌。为专性需氧菌,在普同培养基上不生长,在含有血液的培养基上生长良好,最适温度为37℃。在幼龄培养物中呈球状,在老岭培养物中呈小杆状。无鞭毛,不能运动,不产生芽胞,革兰氏染色阴性,美蓝染色呈两极浓染。本菌的抵抗力较强,在尸体中可存活90多天,但60℃经5~20分即可杀死,3%来苏儿、3%石炭酸以及其他常用消毒药均能很快将其杀死。野兔和野生啮齿动物是本菌的自然储存宿主,也是该病的主要传染源,它们经蜱、蚊等寄生虫通过吸血将病菌传播给家畜和人,此外病菌污染的饲料和饮水亦可经消化道传染动物。各种家畜均能发病,其中绵羊尤其是羔羊发病较为严重,而猪以仔猪较为多见,成年猪多呈隐性感染。春末夏初是本病的高发季节,因为其时野生啮齿动物及其体外寄生虫繁殖最盛。本病的潜伏期一般为l~3天。仔猪出现症状的较为多见。患猪精神沉郁,食欲减退,体温升高至41℃以上,全身衰弱,呼吸困难,呈腹式呼吸,有时咳嗽。病程一般7~10天,多数能耐过,死亡的较少。剖检可见颌下、腮腺淋巴结及其他体表淋巴结肿大、化脓,支气管肺炎,胸膜炎,肝脏实质变性。目前对上述疾病的诊断方法主要有以下几种。
(1)染色镜检:李氏杆菌病可采取肝、脾、脊髓液及脑桥等病料,涂片,革兰氏染色镜检,如发现紫色两端钝圆的细长小杆菌即可确诊。如不能排除猪丹毒时,可将病料作细菌分离培养。坏死杆菌病在病健组织交界处取材,涂片、染色,镜检可见到革兰氏阴性多形杆菌,呈串珠状长丝或细长菌体。土拉杆菌病病料染色镜检,可见革兰氏阴性多形态的细菌。
(2)细菌分离培养:李氏杆菌病病料接种于兔血琼脂平板、0.05%亚碲酸盐胰蛋白胨琼脂平板、麦康凯琼脂平板和1%葡萄糖血清肉汤进行分离培养,在血平板上菌落周围呈b溶血,亚碲酸盐平板上长成圆形、隆起、湿润、黑色的菌落,麦康凯上不生长,肉汤培养呈均匀浑浊,形成颗粒状沉淀。本菌发酵葡萄糖、水杨苷和蕈糖,产酸,接触酶阳性,不产生硫化氢和靛基质,不液化明胶,不还原硝酸盐,M.R试验和V.P.试验阳性。坏死杆菌病取肝、肺、皮肤溃疡灶脓汁接种于0.02%结晶紫或0.01%孔雀绿卵黄培养基,经48~72小时后,本菌长出一种带蓝色的菌落,中央不透明,边缘一圈亮带。土拉杆菌病取感染猪的淋巴结,接种于含有先锋霉素(每毫升40单位)、抗敌素(每毫升100单位)的半胱氨酸葡萄糖、血液琼脂平板上,37℃培养24小时,挑取可疑菌落染色镜检。同时挑取可疑菌落,分别接种于卵黄培养基、费朗西斯培养基和普通培养基上,在前两种培养基中生长,后一种培养基上不生长时判为阳性。
(3)动物实验:李氏杆菌病可点眼接种家兔、小鼠、幼鸽或幼豚鼠,一天后发生结膜炎,不久发生败血症死亡。坏死杆菌病将病料用生理盐水制成悬液,分别给家兔、小鼠皮下注射。家兔耳外侧皮下注射0.5~1毫升,小鼠尾根注射0.2~0.4毫升,被接种动物逐日消瘦,接种局部坏死。家兔注射后2~3天,接种部位形成坏死区,耳下垂,经8~10天死亡。小鼠注射后3天在注射部位发生脓肿,5~6天发生坏死,8~12天死亡。剖检内脏有转移性坏死灶。肝脏涂片镜检或分离培养发现本菌即可确诊。
土拉杆菌病将感染猪的淋巴结制备成悬液,取豚鼠皮下注射0.5毫升,4~10天死亡后,剖检可见肝、脾有多发性坏死灶。用土拉费朗西斯菌素注射病猪尾根皮内0.2毫升,注射后24小时、48小时观察结果。局部发红、肿胀、疼痛者判为阳性。仅有不明显的水肿判为可疑。无任何变化的判为阴性。
(4)聚合酶链式反应(PCR)技术:PCR技术具有高灵敏度、高特异性等优点,已被广泛运用于许多疾病的病原体鉴定和流行病学调查。上述疾病的病原体检测均可使用PCR技术进行检测。
上述检测方法需要专业人员在实验室操作,操作繁琐,检测费时费力;而且需要昂贵的仪器设备,如PCR仪、酶标仪和CO2培养箱等,对非专业人员而言,上述检测方法很难完成。虽然上述方法特异敏感,但每次只能检测一种猪病,且无法实现现场快速检测或诊断。本发明,研究一种简便快速、实时在线,同时检测三种猪病的三联检测试纸,对控制和消灭此类疾病意义重大。
三、发明内容
本发明的目的是为了克服现有技术中检测猪病病原存在的缺点,提供一种特异、敏感、简便快速的呼吸道疾病检测方法,研制出一次可同时检测三种猪细菌性呼吸道疾病的三联检测试纸。
本发明的技术方案是:提供一种猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病三联检测试纸条,该试纸条含有支撑层和吸附层,支撑层为不吸水的薄片层,吸附层附着在支撑层上,吸附层从测试端依次为样品吸附纤维层、金标抗体纤维层、纤维素膜层和手柄端的吸水材料层,在纤维素膜层上设有检测印迹和对照印迹;金标抗体纤维层吸附有纳米级金颗粒标记的抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的三种单克隆抗体,检测印迹用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的配对单抗印制,对照印迹用羊抗或兔抗小鼠IgG的多克隆抗体;或金标抗体纤维层吸附有纳米级金颗粒标记的抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体的多克隆抗体,检测印迹分别用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的单抗制备,对照印迹用金黄色葡萄球菌A蛋白(SPA)或抗猪IgG多抗制备。
检测印迹用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的配对单抗制备即用上述三种病原体特异抗原的配对单抗溶液分别制备;检测印迹用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体的多克隆抗体制备即为用上述三种病原体的多克隆抗体分别制备。
支撑层用不吸水的硬质塑胶片条或硬纸条制成;测试端样品吸附纤维层用玻璃棉制成;金标抗体纤维层用玻璃棉和金标抗体制成,金标抗体可以是单抗或多克隆抗体。
纤维素膜层用硝酸纤维素膜、或纯纤维素膜、或羧化纤维素膜、或聚偏二氟乙烯PVDF纤维素膜制成。
吸水材料层用吸水纸制成。
检测印迹和对照印迹为直线式、或斜线式,纤维素膜层上含有三条检测印迹和一条对照印迹,检测印迹和对照印迹的排列形式为“||||”、“////”、“\\\\”中的任一种。
试纸条吸附层上面含有一层保护层,保护层附着在吸附层上,在测试端样品吸附纤维层、金标抗体纤维层及吸水材料层上覆盖有保护膜,在测试端样品吸附纤维层与金标抗体纤维层交界处对应的保护膜上印制有样品标记线,该标记线偏向测试端样品吸附纤维层一侧处约0.5cm处。
根据需要,选择上述金标抗体纤维层、检测印迹和对照印迹排列形式中一种形式。
本发明的积极有益效果:
1.检测特异性强、敏感性高:本发明检测试纸条以纳米级金颗粒标记高亲和力特异性单克隆抗体或特异性多克隆抗体为基础而制成,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,金颗粒不影响单克隆抗体或多克隆抗体的特异性和结合力,并且具有较高的标记率。本发明检测试纸条具有较高的特异性和敏感性,可检测到纳克级病原体蛋白。
2.操作简便、快速:使用本发明试纸条检测时无需附加任何其它仪器和试剂,只需将其测试端插入待检的样品液中30秒左右,然后在1-5分钟内即可判定检测结果。
3.检测结果直观、准确:本发明试纸条以是否显示棕红色的检测线和对照线作为判定阳性和阴性结果的依据,即只在纤维素膜的对照线印记处显示一条棕红色对照线C,而在检测线印记处无棕红色条带显示,表示被检测的3种猪病均为阴性结果;在纤维素膜的对照线印记处显示一条棕红色对照线C,在检测印迹处出现三条棕红色条带T1、T2、T3(T1为猪李氏杆菌病,T2为猪坏死杆菌病,T3为猪土拉杆菌病),则表示被检测的3种猪病均为阳性结果;在纤维素膜上显示一条棕红色对照线和三条棕红色检测线中的任一条或任两条,则表示在被检测3种猪病中的任一种或两种为阳性结果。无论阳性结果或阴性结果对照线C均应显示,当对照线C不显示时,说明试纸条失效。
4.检测费用降低:使用本发明检测试纸条,不需其它仪器及试剂,节省了仪器、设备和附加试剂费用;一条试纸一次可以检测3种猪病,非专业人员也可随时实时在线检测,无需支付专家诊断检查费及其相关费用,可极大的降低检测成本的投入,降低检测费用。
5.使用范围广:本发明检测试纸条操作简单,即“傻瓜式”操作,而且携带方便、易保存,可满足不同单位和不同层次人员的需要,包括专业化验、海关检疫、卫生防疫、质量监测、畜产品加工、集约化养殖到个体养殖等,具有广阔的市场前景和社会效益。
四、附图说明:
图1一种猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病三联检测试纸条的侧视结构示意图
图2一种猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病三联检测试纸条的俯视结构示意图
五、具体实施方式:
以下实施例仅为了进一步说明本发明,并不限制本发明的内容。猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病三联检测试纸条的制备,需要制备抗三种病原体特异抗原的单克隆抗体和多克隆抗体,用于制备检测印迹和金标抗体纤维层;同时需要制备羊或兔抗鼠IgG抗体,或羊或兔抗猪IgG抗体,用于制备对照印迹。
1.羊(兔)抗鼠或猪IgG抗体的制备:
以饱和硫酸铵法提取小鼠或猪血清中的IgG,取1份血清加2份PBS液(pH7.2)混匀,加等体积饱和硫酸铵液混匀,置4℃冰箱内2h,在4℃、10000r/min离心15min,弃上清液;以适量PBS液(pH7.2)溶解沉淀,加饱和硫酸铵液至其最终浓度为33%,置4℃冰箱内2h,在4℃、10000r/min条件下离心15min,弃上清液,以少量PBS液(pH7.2)溶解沉淀,置4℃冰箱内用PBS液(pH7.2)过夜透析,换液2~3次,在4℃、10000r/min条件下离心15min,收集上清液,以紫外分光光度计测定其蛋白浓度。以50μg~100μg(IgG)/kg体重经皮下或肌肉注射抗体阴性健康羊或家兔3~4次,末次免疫20天后,静脉采血,以ELISA测定其血清抗体效价在1:2000以上,心脏采血或颈动脉放血,收集其高免血清,以饱和硫酸铵法提取羊(兔)抗小鼠或猪的IgG(其提取方法与上述提取小鼠血清IgG相同,不再重述),用于制备本发明试纸条的对照印迹。
2.猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原单克隆抗体(Mi)的制备:
每只用50μg~100μg病原体特异抗原免疫Balb/c系小鼠三次,每次间隔15~30d;第三次加强免疫后3~4d,将免疫小鼠眼球放血,拉颈致死,用75%酒精浸泡5~10min,无菌取其脾脏,剪碎并经100目尼龙网过滤,1000r/min离心10min,收集脾细胞;将1×108个脾细胞与2~5×107个NS0骨髓瘤细胞混合,1000r/min离心10min弃上清,将含有沉淀细胞的离心管置于37℃的水中,并缓缓加入0.7~1ml40%~50%PEG4000(pH8.5~9.0)作用1min,然后缓慢加入无血清1640培养基15ml,以终止PEG的作用,37℃水浴5~10min,1000r/min离心10min弃上清,将细胞沉淀重悬于HAT选择培养基中,并加入
96孔培养板(100μl~200μl/孔),置于37℃5%CO2培养箱中培养。培养7~10d后,以5μg~10μg/ml的纯化的病原体特异抗原包被96孔酶标板,以酶联免疫吸附试验(ELISA)检测杂交瘤的培养上清,挑取强阳性细胞克隆(OD450≥0.5),进行连续三次的有限稀释法克隆化,获得阳性杂交瘤细胞株,其染色体数为92~98,其分泌的单克隆抗体(M1,M2或M3),能够特异识别三种不同的病原体,而不与其它猪的病原体发生交叉反应,亲和力常数达109 ~10,轻链亚型为к或λ,重链亚型为IgG1、IgG2a、IgG2b、IgG3;获得的配对单克隆抗体,用于制金标单抗体玻璃棉或检测印迹。
3.金标单抗玻璃棉的制备:
利用柠檬酸钠还原法制备纳米级金颗粒:即在50~100ml沸腾的0.01~0.05%氯金酸水溶液中加入2~4ml的0.5~2%柠檬酸三钠溶液,获得直径15nm左右的纳米级金颗粒。以0.1mol/L的K2CO3调金颗粒溶液的pH至8.5~9.5,以1:1000~1300的标记比将待标记的单克隆抗体(M1,M2或M3)加入pH8.5~9.5的金溶胶中,标记10min后,加20%PEG10000至最终浓度为0.05%,4℃、1500~3000r/min离心20min,除去未结合的金颗粒颗粒,4℃、15000r/min离心1h,弃上清,获金标抗体混合物后,用丙烯葡聚糖S-400柱层析,分离纯化金标抗体,分别获得M1、M2、M3的金标抗体。将1:100~500稀释的三种金标抗体,吸附于精制玻璃棉中,4℃低温真空干燥,制备金标单克隆抗体玻璃棉。
4.病原体特异抗原多克隆抗体(Ci)的制备:
病原体特异抗原多克隆抗体(Ci)的制备。分别采用国家批准的上述三种猪病的灭活疫苗、弱毒疫苗或标准抗原,多次免疫接种抗体阴性健康猪。末次免疫20天后静脉采血,以ELISA测定其血清抗体效价在1:2000以上,心脏采血或颈动脉放血,收集其高免血清,以饱和硫酸铵法提取血清中IgG抗体(方法与小鼠血清IgG的提取相同,不再重述)。
金标多抗和金标多抗玻璃棉的制备,与金标单抗玻璃棉的制备方法相同,不再重述。
详见具体实施方式中的内容3。
5.本发明检测试纸条检测原理
当本发明检测试纸条测试端插入待检样品溶液后,待检溶液通过虹吸带动待检病原体进入金标抗体纤维层,并与其中的金标抗体(Mi或Ci)一起沿硝酸纤维素膜向手柄端扩散,最终渗入手柄端吸水材料层,扩散过程中金标抗体能够与相应的待检病原体结合,结合的金标抗体的病原体能够被纤维素膜上检测印迹的配对单抗或多抗拦截,当样品液中含有被检病原体时,则出现1~3条棕红色的检测线;羊或兔抗鼠或抗猪IgG则可与相应的金标单抗或多抗结合,出现1条棕红色对照线。当待检样品液中没有上述病原体时,试纸条只显示出一条棕红色对照线;当纤维素膜上没有对照线显示时,则表明试纸条已失效。
6.本发明检测试纸条的检测操作方法
(1)检测样品的处理:取病猪病变组织,1:1~5加入生理盐水并用剪刀剪碎,浸出液为待检样品,病猪全血或血清加入生理盐水1:1~5稀释后为待检样品。
(2)检测操作:将本发明检测试纸条样品端插入待检样品液中,插入深度不超过标记线9,约30秒后取出试纸条,水平放置约1~5分钟,同时观察结果。
(3)结果判定:如果在检测试纸条纤维素膜上只显示出一条棕红色对照线C,表示检测结果为阴性,说明在被检样品中不含上述3种病原体;如果检测试纸条上的纤维素膜出现对照线C,检测印迹处出现T1或T2或T3检测线,表示检测结果为阳性,即在待检样品中含有猪李氏杆菌病病原体或猪坏死杆菌病病原体或猪土拉杆菌病病原体;如果检测印迹处T1或T2或T3同时出现,表示在待检样品中存在上述3种病原体;如果纤维素膜上没有任何棕红色印迹显示,则表明试纸条已失效。
实施例一:猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病三联检测试纸条
参见图1和图2,图中1为支撑层,用硬质塑胶薄片条制成,2为测试端的样品吸附纤维层,用玻璃棉制成,3为金标抗体纤维层,吸附有纳米级金颗粒标记的抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体的三种单克隆抗体的玻璃棉,根据上述具体实施方式3中所述的制备方法制备其金标单抗玻璃棉,4为纤维素膜层,采用硝酸纤维素膜制成,5为吸水材料层,用吸水纸制成,将编号2、3、4、5各层从左端测试端至右粘贴在硬质塑胶薄片条1上,彼此之间交界处互相交叉重叠。在硝酸纤维素膜层4上,6为分别用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体的配对单抗溶液印制的检测印迹T1、T2、T3,7为用羊或兔抗鼠IgG溶液印制的对照印迹C,检测印迹和对照印迹为直线式、或斜线式,两种印迹带排列形成的组合形式为“||||”、“////”、“\\\\”中的任一种。8-1为覆盖在测试端样品吸附纤维层2和金标抗体纤维层3上面的白色保护膜,在2和3交界处对应保护膜8-1位置上偏向于样品吸附纤维层2一侧0.5cm处印有标记线9,9的右端印有箭头及max字样,吸水材料层5(手柄端)上覆盖有其它颜色(如黄色)保护膜8-2。
待测样品溶液的制备及检测操作步骤,与具体实施方式6中的检测操作方法相同,不再重述。
实施例二:猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病三联检测试纸条,与实施例一基本相同,不同之处在于:
金标抗体纤维层3用吸附有金颗粒标记的抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病的三种多克隆抗体的玻璃棉制成,根据上述具体实施方式3中所述的制备方法制备其金标多克隆抗体玻璃棉;在硝酸纤维素膜层4上,6为分别用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体的单抗溶液印制的检测印迹T1、T2、T3,7为用羊或兔抗猪的IgG溶液印制对照印迹C,两种印迹带排列形成的组合形式为“||||”、“////”、“\\\\”中的任一种。其它包括检测样品制备、操作方法和结果判定等均与具体实施方式6中的操作方法相同,不再重述。
Claims (7)
1.一种检测猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病的三联检测试纸条,该试纸条含有支撑层和吸附层,支撑层为不吸水的薄片层,吸附层附着在支撑层上,吸附层从测试端依次为样品吸附纤维层、金标抗体纤维层、纤维素膜层和手柄端的吸水材料层,在纤维素膜层上制备有检测印迹和对照印迹,其特征是金标抗体纤维层吸附有纳米级金颗粒标记的抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的三种单克隆抗体,检测印迹用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的配对单抗或多克隆抗体印制,对照印迹用羊或兔抗小鼠IgG的多克隆抗体或金黄色葡萄球菌A蛋白印制;或金标抗体纤维层吸附有纳米级金颗粒标记的抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体的多克隆抗体,检测印迹分别用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的单抗制备,对照印迹用金黄色葡萄球菌A蛋白或抗猪IgG多抗制备;
其中,抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的三种单克隆抗体的制备方法如下:
用50μg~100μg病原体特异抗原免疫Balb/c系小鼠三次,每次间隔15~30d;第三次加强免疫后3~4d,将免疫小鼠眼球放血,拉颈致死,用75%酒精浸泡5~10min,无菌条件下取其脾脏,剪碎并经100目尼龙网过滤,1000r/min离心10min,收集脾细胞;将1×108个脾细胞与2~5×107个NS0骨髓瘤细胞混合,1000r/min离心10min弃上清,将含有沉淀细胞的离心管置于37℃的水中,并缓缓加入0.7~1ml40%~50%PEG4000作用1min,所述PEG4000的pH为8.5~9.0,然后缓慢加入无血清1640培养基15ml,以终止PEG的作用,37℃水浴5~10min,1000r/min离心10min弃上清,将细胞沉淀重悬于HAT选择培养基中,并以100μl~200μl/孔加入96孔培养板,置于37℃5%CO2培养箱中培养;培养7~10d后,以5μg~10μg/ml的纯化的病原体特异抗原包被96孔酶标板,以酶联免疫吸附试验检测杂交瘤的培养上清,挑取OD450≥0.5的强阳性细胞克隆,进行连续三次的有限稀释法克隆化,获得阳性杂交瘤细胞株,其染色体数为92~98,其分泌的单克隆抗体能够特异识别相应的病原体,而不与其它猪的病原体发生交叉反应,亲和力常数达109~10,轻链亚型为к或λ,重链亚型为IgG1、IgG2a、IgG2b或IgG3。
2.根据权利要求1所述的试纸条,其特征是检测印迹用抗猪李氏杆菌病、猪坏死杆菌病及猪土拉杆菌病病原体特异抗原的配对单抗制备即用上述三种病原体特异抗原的配对单抗溶液分别制备。
3.根据权利要求1所述的试纸条,其特征是支撑层用不吸水的硬质塑胶片条或硬纸条制成;测试端样品吸附纤维层用玻璃棉制成;金标抗体纤维层用玻璃棉和金标抗体制成,金标抗体是单抗或多克隆抗体。
4.根据权利要求1所述的试纸条,其特征是纤维素膜层用硝酸纤维素膜、或纯纤维素膜、或羧化纤维素膜制成。
5.根据权利要求1所述的试纸条,其特征是吸水材料层用吸水纸制成。
6.根据权利要求1所述的试纸条,其特征是检测印迹和对照印迹为直线式、或斜线式,纤维素膜层上含有三条检测印迹和一条对照印迹,检测印迹和对照印迹的排列形式为“||||”、“////”、“\\\\”中的任一种。
7.根据权利要求1所述的试纸条,其特征是在测试端样品吸附纤维层、金标抗体纤维层及吸水材料层上覆盖有保护膜,在测试端样品吸附纤维层与金标抗体纤维层交界处对应的保护膜上印制有样品标记线,该标记线偏向测试端样品吸附纤维层一侧约0.5cm处。
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