CN103869071A - 单克隆抗体间接免疫荧光检测附红细胞体病的方法 - Google Patents
单克隆抗体间接免疫荧光检测附红细胞体病的方法 Download PDFInfo
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Abstract
本发明公开了一种单克隆抗体间接免疫荧光检测附红细胞体病的方法,包括标准阳性对照和阴性对照的设立,抗原包被,荧光二抗的稀释,IFA测定等步骤。采用的抗体是抗猪附红细胞体的单克隆抗体,相对于IHA、ELISA等其他检测方法所用的一般的抗体而言,其特异性高、敏感性强,准确性可靠性大;能快速、简便、可靠、准确的检测出样品中的附红细胞体,也可用于附红细胞体病的流行病学调查和疗效检测。
Description
技术领域
本发明属于生物工程检测技术领域,具体涉及单克隆抗体间接免疫荧光检测附红细胞体病的方法。
背景技术
附红细胞体病(Eperythrozoonsis)是由附红细胞体(Eperythmzoon,EH)寄生于人、动物红细胞表面、血浆及骨髓内,引起的一种以贫血、黄疸、发热等为主要临床症状的人畜共患病。属于立克次体目(Rickettsiales)、无浆体科(Anaplasmata-ceae)、附红细胞体属(Eperythrozoon)。多在红细胞表面单个或成团,呈链状或鳞片状,少量游离。猪附红细胞体一般呈环形,直径0.8-2.5um,也有球状、杆状。对干燥和化学药物比较敏感,0.5%石炭酸于37℃经3h可将其杀死,一般常用浓度的消毒药在几分钟内即可使其死亡,对低温冷冻的抵抗力较强,可存活数年之久。附红细胞体对宿主的选择并不严格,人、牛、猪、羊等多种动物均可感染,且感染率比较高。有人做过调查,各种阶段猪的感染率达80-90%;人的感染阳性率可达86%;而鸡的阳性率更高,可达90%,但除了猪之外的其它动物发病率不高。多数隐性,少数发病(受应激因素刺激),潜伏期2-45d,因动物种类不同发病症状也不同,发热,食欲不振,精神萎顿,黏膜黄染,贫血,背腰及四肢末梢瘀血,淋巴结肿大,心悸及呼吸加快;腹泻,生殖力下降,毛质下降,红细胞数减少,血红素、血浆白蛋白、球蛋白均下降,淋巴细胞及单核细胞上升,血红蛋白尿病程长短不一,短者几天,长者数年,严重者死亡。
该病最早发现于1928年,直到1950年确定猪的黄疸性贫血是由附红细胞体所引起,才逐渐被重视起来。我国于1981年首先在家兔中发现附红细胞体,相继在牛、羊、猪等家畜中查到附红细胞体,以后在人群中也证实了附红细胞体感染的存在。该病不仅导致畜产品质量和产量降低,动物生育力下降,而且还可导致动物发生严重的临床症状甚至死亡,阻碍了畜牧业的发展,造成了重大的经济损失。目前对该病的诊断仍多采用镜检的方法,但由于一些人为的因素及该病多呈混合感染,常会发生误诊。近年来发展起来的分子生物学检测方法如PCR(聚合酶链式反应)、核酸杂交等已开始应用到该病的检测诊断中,但特异敏感的检测方法急需完善,假阴性和假阳性的几率非常大。所以,目前急需一种特异性强、敏感性高的检测方法用于附红细胞体病的诊断。
发明内容
本发明的目的在于,克服现有技术存在的缺点,提供一种单克隆抗体间接免疫荧光检测附红细胞体病的方法,快速、简便、可靠、准确的检测出附红细胞体病,以满足畜牧业生产和医学诊断的需求。
本发明提供的单克隆抗体间接免疫荧光检测附红细胞体病的方法,包括以下步骤:
(1)标准阳性对照和阴性对照的设立,将自然感染率达90%以上,且经瑞氏、姬姆萨和吖啶橙染色检测均为阳性的猪血作为标准阳性抗原对照;将来自西藏地区且经压片、瑞氏、姬姆萨和吖啶橙染色及PCR检测均为阴性的猪血作为阴性对照;
(2)抗原包被,取标准阳性和阴性血样涂片,晾干,并设空白对照;
(3)荧光二抗的稀释,分别用0.01% 的伊文思蓝和0.01 mol/L PBS稀释FITC标记的山羊抗鼠IgG抗体;
(4)IFA测定,将已包被好的两组标准阳性和阴性对照的玻片,用冷丙酮和甲醛等比例混合液固定15 min,室温下干燥,加入相应编号的抗猪附红细胞体的单克隆抗体20 L,置湿盒中37℃下作用30 min,用PBS轻轻冲洗,然后浸泡于PBS盒中,于电动摇床上轻微摇动,连续洗涤3次,待免疫荧光片干燥后将其中一组标准阳性和阴性抗原及空白对照玻片中加入10 L用0.01%伊文思蓝稀释的荧光抗体;另一组加入10L用0.01mol/LPBS稀释的荧光抗体,置湿盒中37℃下作用30 min,用PBS轻轻冲洗,然后浸泡于PBS盒中,于电动摇床上轻微摇动,连续洗涤3次,待干燥后滴加甘油-PBS缓冲液,覆加盖玻片,置荧光显微镜下观察;
所述步骤(1)中所用的瑞氏、姬姆萨和吖啶橙染色方法步骤如下:
(1)瑞氏染色,取一洁净载玻片,以其右端1/3蘸血一滴,作血涂片,自然晾干后滴加瑞氏染液1min,加等量0.01mol/L PBS(PH6.4)混匀,染色3-5min,水洗,室温干燥后镜检;
(2)姬姆萨染色,作血涂片,自然晾干后用甲醇固定2-3min,加姬姆萨染液15-30min,水洗,室温干燥后镜检;
(3)吖啶橙染色,做血涂片,自然晾干后用95%乙醇固定15-30min,滤纸吸干,1%醋酸作用30s,PBS清洗1min,于0.01%吖啶橙染液中染色1-5min,PBS清洗1min,用0.1mol/L CaCl2分色0.5-2min,PBS清洗3次,每次数秒,甘油和PBS等比例混合封片,立刻于荧光显微镜下观察。
本发明提供的单克隆抗体间接免疫荧光检测附红细胞体病的方法,其有益效果在于:提供了一种检测附红细胞体病的标准方法,采用的抗体是抗猪附红细胞体的单克隆抗体,相对于IHA、ELISA等其他检测方法所用的一般的抗体而言,其特异性高、敏感性强,准确性可靠性大;能快速、简便、可靠、准确的检测出样品中的附红细胞体,也可用于附红细胞体病的流行病学调查和疗效检测。
具体实施方式
下面结合一个实施例,对本发明提供的单克隆抗体间接免疫荧光检测附红细胞体病的方法进行详细的说明。
实施例
本实施例的单克隆抗体间接免疫荧光检测附红细胞体病的方法,包括以下步骤:
(1)标准阳性对照和阴性对照的设立,将自然感染率达90%以上,且经瑞氏、姬姆萨和吖啶橙染色检测均为阳性的猪血作为标准阳性抗原对照;将来自西藏地区且经压片、瑞氏、姬姆萨和吖啶橙染色及PCR检测均为阴性的猪血作为阴性对照;
(2)抗原包被,取标准阳性和阴性血样涂片,晾干,并设空白对照;
(3)荧光二抗的稀释,分别用0.01% 的伊文思蓝和0.01 mol/L PBS稀释FITC标记的山羊抗鼠IgG抗体;
(4)IFA测定,将已包被好的两组标准阳性和阴性对照的玻片,用冷丙酮和甲醛等比例混合液固定15 min,室温下干燥,加入相应编号的抗猪附红细胞体的单克隆抗体20 L,置湿盒中37℃下作用30 min,用PBS轻轻冲洗,然后浸泡于PBS盒中,于电动摇床上轻微摇动,连续洗涤3次,待免疫荧光片干燥后将其中一组标准阳性和阴性抗原及空白对照玻片中加入10 L用0.01%伊文思蓝稀释的荧光抗体;另一组加入10L用0.01mol/LPBS稀释的荧光抗体,置湿盒中37℃下作用30 min,用PBS轻轻冲洗,然后浸泡于PBS盒中,于电动摇床上轻微摇动,连续洗涤3次,待干燥后滴加甘油-PBS缓冲液,覆加盖玻片,置荧光显微镜下观察;
所述步骤(1)中所用的瑞氏、姬姆萨和吖啶橙染色方法步骤如下:
(1)瑞氏染色,取一洁净载玻片,以其右端1/3蘸血一滴,作血涂片,自然晾干后滴加瑞氏染液1min,加等量0.01mol/L PBS(PH6.4)混匀,染色3-5min,水洗,室温干燥后镜检;
(2)姬姆萨染色,作血涂片,自然晾干后用甲醇固定2-3min,加姬姆萨染液15-30min,水洗,室温干燥后镜检;
(3)吖啶橙染色,做血涂片,自然晾干后用95%乙醇固定15-30min,滤纸吸干,1%醋酸作用30s,PBS清洗1min,于0.01%吖啶橙染液中染色1-5min,PBS清洗1min,用0.1mol/L CaCl2分色0.5-2min,PBS清洗3次,每次数秒,甘油和PBS等比例混合封片,立刻于荧光显微镜下观察。
Claims (2)
1.一种单克隆抗体间接免疫荧光检测附红细胞体病的方法,其特征在于:包括以下步骤:
(1)标准阳性对照和阴性对照的设立,将自然感染率达90%以上,且经瑞氏、姬姆萨和吖啶橙染色检测均为阳性的猪血作为标准阳性抗原对照;将来自西藏地区且经压片、瑞氏、姬姆萨和吖啶橙染色及PCR检测均为阴性的猪血作为阴性对照;
(2)抗原包被,取标准阳性和阴性血样涂片,晾干,并设空白对照;
(3)荧光二抗的稀释,分别用0.01% 的伊文思蓝和0.01 mol/L PBS稀释FITC标记的山羊抗鼠IgG抗体;
(4)IFA测定,将已包被好的两组标准阳性和阴性对照的玻片,用冷丙酮和甲醛等比例混合液固定15 min,室温下干燥,加入相应编号的抗猪附红细胞体的单克隆抗体20 L,置湿盒中37℃下作用30 min,用PBS轻轻冲洗,然后浸泡于PBS盒中,于电动摇床上轻微摇动,连续洗涤3次,待免疫荧光片干燥后将其中一组标准阳性和阴性抗原及空白对照玻片中加入10 L用0.01%伊文思蓝稀释的荧光抗体;另一组加入10L用0.01mol/LPBS稀释的荧光抗体,置湿盒中37℃下作用30 min,用PBS轻轻冲洗,然后浸泡于PBS盒中,于电动摇床上轻微摇动,连续洗涤3次,待干燥后滴加甘油-PBS缓冲液,覆加盖玻片,置荧光显微镜下观察。
2.根据权利要求1所述的单克隆抗体间接免疫荧光检测附红细胞体病的方法,其特征在于:所述步骤(1)中所用的瑞氏、姬姆萨和吖啶橙染色方法步骤如下:
(1)瑞氏染色,取一洁净载玻片,以其右端1/3蘸血一滴,作血涂片,自然晾干后滴加瑞氏染液1min,加等量0.01mol/L PBS(PH6.4)混匀,染色3-5min,水洗,室温干燥后镜检;
(2)姬姆萨染色,作血涂片,自然晾干后用甲醇固定2-3min,加姬姆萨染液15-30min,水洗,室温干燥后镜检;
(3)吖啶橙染色,做血涂片,自然晾干后用95%乙醇固定15-30min,滤纸吸干,1%醋酸作用30s,PBS清洗1min,于0.01%吖啶橙染液中染色1-5min,PBS清洗1min,用0.1mol/L CaCl2分色0.5-2min,PBS清洗3次,每次数秒,甘油和PBS等比例混合封片,立刻于荧光显微镜下观察。
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CN107177524A (zh) * | 2017-04-28 | 2017-09-19 | 内蒙古医科大学 | 一种人附红细胞体体外培养方法 |
CN107589117A (zh) * | 2017-08-22 | 2018-01-16 | 贵州省畜牧兽医研究所 | 一种猪附红细胞体病快速镜检诊断试剂盒及其制备方法 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107177524A (zh) * | 2017-04-28 | 2017-09-19 | 内蒙古医科大学 | 一种人附红细胞体体外培养方法 |
CN107589117A (zh) * | 2017-08-22 | 2018-01-16 | 贵州省畜牧兽医研究所 | 一种猪附红细胞体病快速镜检诊断试剂盒及其制备方法 |
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