CN103941002B - 猪丹毒杆菌抗原快速检测试纸条 - Google Patents
猪丹毒杆菌抗原快速检测试纸条 Download PDFInfo
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Abstract
本发明涉及一种猪丹毒杆菌抗原检测试剂显示的器具,特别是涉及一种猪丹毒杆菌抗原快速诊断试纸条,试纸条含有支撑层、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,猪丹毒杆菌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;分别用猪丹毒杆菌抗原配对单抗或多抗或单抗溶液在纤维层上印制检测印迹“<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印制对照印迹“<b>|</b>”、“<b>/</b>”或“<b>\</b>”。该检测试纸条,特异性强,敏感性高,检测结果显示形象、直观、准确,无需仪器设备,无需专业检测人员,费用低,操作简便、快速,可大大降低劳动强度,缩短检测时间,能在饲养场,肉类加工厂,出入境检验检疫局等场所进行现场检测,易于推广应用。
Description
一、技术领域
本发明是一种涉及猪丹毒杆菌抗原的检测试剂显示器具,特别是涉及一种可检测猪丹毒杆菌抗原的检测试纸条。
二、技术背景
猪丹毒是由猪丹毒杆菌引起猪的一种急性、热性传染病。不同年龄的猪均可感染,但主要侵害3~12月龄的架子猪,哺乳仔猪和产子母猪很少发生。人感染发病时称为类丹毒。本病虽然一年四季均可发生,但在北方地区以夏季炎热、多雨季节流行最盛,而在南方地区则在冬春季节流行。常为散发性或地方流行传染,有时爆发流行。主要由污染的饲料、土壤经消化道感染,创伤皮肤也可感染。该病流行于世界各地,对养猪业危害很大。病猪、带菌猪、及带菌禽类是该病的主要传染源,潜伏期3~5天,其发病特征为急性病例呈败血型,开始发病时1~2头猪无任何症状突然死亡,随后出现较多的发病或死亡病猪,体温升高42℃以上。食欲废绝,行走摇摆,呼吸困难,粘膜发绀,发病几天后,胸、腹、四肢内侧及耳部皮肤出现大小不等的红斑,指压退色,病末期心脏衰弱、体温下降、虚脱死亡。剖检皮肤充血呈弥漫性红色。脾脏充血、肿大、柔软,呈暗红褐色。肾脏黄褐色或暗红色,充血肿大。肝淤血、棕红色。胃粘膜、十二指肠、空肠粘膜肿胀,大肠无明显变化。亚急性病例,在皮肤上发生大小不等、形状不一的紫红色疹块,俗称打火印。剖检病死猪除有特征性的皮肤疹块外,其脾、肾也表现败血型变化。慢性大多由急性、亚急性转变而来,主要表现关节炎和心内膜炎,剖检关节肿大、关节腔内淡黄色液体增多,关节囊增厚,关节变形。心内膜炎型常见于二尖瓣形成颗粒状增生物,外观似菜花样病变。常用检测方法如下。
(1)显微镜检查:将病料最好是肝、肾涂片(心内膜疣状赘生物可表面触片),自然干燥,用甲醇固定2~5分后,用美蓝或复红也可用姬姆萨氏或瑞特氏染色法及革兰氏染色法染色后镜检。本菌在镜下呈瘦长、正直或稍弯曲、纤细的杆菌。并呈散在、单个、成对或小堆状或簇集于白细胞中。从心内膜赘生物制成的涂片,常可见有弯曲、长短不等、丝状菌体,并呈乱发状。本菌为革兰氏阳性小杆菌;不产生芽胞;无鞭毛,不能运动。
(2)细菌培养:①直接分离培养对未被污染的新鲜病料可直接接种于血液琼脂或血清琼脂培养基,置37℃培养24~48小时后,观察结果。在血清琼脂上长出针尖大、透明、灰白色、圆形、微隆起的露珠状小菌落。在血液琼脂培养基上生长的菌落,周围有绿色的狭窄溶血环。如被检材料已经腐败或被污染,可在上述培养基中加入抗生素(100微克/毫升新霉素或400微克/毫升卡那霉素或加入叠氮钠和结晶紫各0.01%)抑制其它革兰氏阴性菌生长。②增菌培养如病料中病原菌少时,应先作增菌培养,即取病料一铂耳,放入普通肉汤或血清肉汤(后者较好)中,培养24小时后观察结果。
猪丹毒在肉场中培养24小时后,肉汤呈均匀一致的轻微混浊,管底有少量沉淀,振荡时沉淀物呈小絮片状浮起,无菌膜及附着于管壁的环状物;老龄培养时,多呈絮块或絮条样生长,并常悬浮于试管中央或沉于管底。
(3)动物试验:当病料中含菌量极少,或已被污染,作细菌分离诊断有困难时,可接种小动物作为辅助诊断,动物试验也是分离鉴定本菌的一个方面。其方法如下:将病料(疹块部渗出液或血液、脾、肝、肾等)或纯培养物接种于鸽子、小白鼠和豚鼠。先将病料磨碎,用灭菌生理盐水作5~10倍稀释制成悬液。鸽于胸肌接种0.5~l毫升,小白鼠皮下接种0.2毫升,豚鼠皮下或腹腔接种0.5~1毫升。若为肉汤培养物可直接接种。固体培养基上的菌落,需先用灭菌生理盐水洗下,制成菌液再接种。接种后1~4日,鸽子翅、腿麻痹,精神萎顿,头缩羽乱,不吃而死亡。小白鼠出现精神萎顿、背拱、毛乱、闭眼、不吃,3~7天死亡。死亡的鸽子和小白鼠可见脾脏肿大,肺和肝充血,肝有时可见小点坏死。取心血、肾、脾等,涂片镜检或分离培养,均可见有多量猪丹毒杆菌。豚鼠对猪丹毒杆菌有很强的抵抗力,接种后常不表现任何症状,仍健康存活。
(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,PCR方法已被广泛运用于猪丹毒杆菌的鉴定和流行病学调查。此外,还建立了检测猪丹毒杆菌的ELISA方法。
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、实时在线检测试纸,对控制和消灭此疾病意义重大。
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、简便快速的猪丹毒杆菌检测方法,研制出检测猪丹毒杆菌抗原的检测试纸条。
本发明的技术方案是:提供一种猪丹毒杆菌抗原的检测试纸条,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丹毒杆菌抗原的单克隆抗体,检测印迹用抗猪丹毒杆菌抗原的配对单抗印制,对照印迹用羊或兔抗小鼠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丹毒杆菌抗原的多克隆抗体,检测印迹用抗猪丹毒杆菌抗原的单抗制备,对照印迹用金黄色葡萄球菌A蛋白(SPA)或抗猪IgG多抗制备。
检测印迹用抗猪丹毒杆菌抗原的配对单抗制备即用猪丹毒杆菌抗原的配对单抗溶液制备;检测印迹用抗猪丹毒杆菌抗原的多克隆抗体制备即为用猪丹毒杆菌抗原的多克隆抗体制备。
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,纤维素膜层上含有一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,表示被检测的猪丹毒杆菌抗原为阴性结果;在纤维素膜的对照线印记处显示一条棕红色对照线C,在检测印迹处显示一条棕红色条带T,则表示被检测的猪丹毒杆菌抗原为阳性结果。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;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1一种猪丹毒杆菌抗原的检测试纸条的侧视结构示意图
图2一种猪丹毒杆菌抗原的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。猪丹毒杆菌抗原检测试纸条的制备,需要制备抗猪丹毒杆菌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,羊或兔抗猪IgG抗体,用于制备对照印迹。
1.羊(兔)抗鼠或抗猪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.猪丹毒杆菌抗原单克隆抗体(Mi)的制备:
每只用50μg~100μg猪丹毒杆菌抗原抗原免疫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,能够特异识别猪丹毒杆菌抗原,而不与其它毒素发生交叉反应,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,获得的金标抗体。将1:100~500稀释的金标抗体,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.猪丹毒杆菌抗原多克隆抗体(Ci)的制备:
猪丹毒杆菌抗原多克隆抗体(Ci)的制备。采用猪丹毒杆菌抗原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猪丹毒杆菌抗原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猪丹毒杆菌抗原结合,结合金标抗体的猪丹毒杆菌抗原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猪丹毒杆菌抗原时,则出现一条棕红色的检测线;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。当待检样品液中不含猪丹毒杆菌抗原时,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猪丹毒杆菌抗原;如果检测试纸条上的纤维素膜出现对照线C,检测印迹处出现一条检测线,表示检测结果为阳性,即在待检样品中含有猪丹毒杆菌抗原;如果纤维素膜上没有对照线C显示,则表明试纸条已失效。
实施例一:猪丹毒杆菌抗原的检测试纸条
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丹毒杆菌抗原的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为用抗猪丹毒杆菌抗原的配对单抗溶液印制的检测印迹T,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:猪丹毒杆菌抗原的检测试纸条,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丹毒杆菌抗原的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为用抗猪丹毒杆菌抗原的单抗溶液印制的检测印迹T,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)
1.一种检测猪丹毒杆菌抗原的检测试纸条,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丹毒杆菌抗原的单克隆抗体,检测印迹用抗猪丹毒杆菌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丹毒杆菌抗原的多克隆抗体,检测印迹用抗猪丹毒杆菌抗原的单抗制备,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,抗猪丹毒杆菌抗原的单克隆抗体的制备方法如下:
用50μg~100μg猪丹毒杆菌抗原免疫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猪丹毒杆菌抗原,而不与其它毒素发生交叉反应,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丹毒杆菌抗原的配对单抗制备即用抗猪丹毒杆菌抗原的配对单抗溶液制备。
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,纤维素膜层上含有一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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