CN110361530A - 一种快速检测菌体种类或抗体的方法 - Google Patents
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Abstract
本发明涉及一种快速检测菌体种类或抗体的方法，属于生物医药领域。本方法主要通过菌体与一抗和二抗三者发生结合反应或者是菌体与标记抗体二者直接发生结合反应，经离心或过滤得到结合物，然后通过底物颜色对菌体种类或抗体进行鉴别。该方法是利用了免疫共沉淀的原理同时又结合了酶联免疫的底物显色的方法形成了一种新型的鉴别菌体种类或抗体的方法。该方法相对于目前检测菌体常用的玻片凝集试验、酶联免疫吸附反应法、胶体金技术和分子生物学等方法具有操作简单、成本低廉、灵敏度高、快速高效和可批量筛查的优点，今后具有广阔的应用前景。
Description
技术领域
本发明属于生物医药领域，尤其涉及一种简单快速鉴定菌体类型或抗体的方法。
背景技术
细菌与人和动物的关系密切。益生菌对于维护机体健康非常重要，目前在人医和兽医临床上广泛应用，常用的益生菌有乳酸菌、芽孢菌和酵母菌等；而致病菌会对机体会造成伤害，如有害的沙门氏菌、大肠杆菌、巴氏杆菌等。细菌涉及到人们生活中的诸多方面，如日常保健、食品加工、疾病诊断等，因此对于细菌的快速鉴别检测非常重要，如益生菌的快速检测有利于相关产品的合格性评价，而有害菌的快速检测有利于疾病的及时、精准治疗，以免延误时间，加重病情。目前对于细菌性疾病诊断主要方法有细菌培养、玻片凝集反应、生化实验、胶体金、酶联免疫和分子生物学实验等方法。这些方法中常规的细菌培养，耗时长，操作繁琐，不宜用于细菌感染的快速诊断；玻片凝集反应法，利用肉眼观察判断有无凝集反应，虽操作简单，但准确性低；生化实验操作较繁琐，培养细菌时间长；胶体金虽然检测较快速，但成本高而且不适合大量筛查；酶联免疫成本高，操作时间长；分子生物学虽准确性高，但需要具备相关技术和设备等。总之，上述检测方法均有优势和缺点，适合不同的场所和需求。
本发明涉及一种菌体种类鉴别方法，将免疫共沉淀与酶联免疫显色反应巧妙结合，发明了一种新型的鉴别菌体种类的方法。可以将该方法称为菌体的“免疫沉淀-酶联显色法”。
发明内容
一种菌体或抗体鉴别检测的方法，该方法将菌体与一抗、二抗混合在一起进行结合反应，然后离心或过滤得沉淀，再加入显色底物，通过颜色反应鉴别菌体或抗体的种类。这种情况适合于那些与二抗无法直接结合的菌种。
也可以是菌体、抗血清和标记二抗混合进行结合，离心或过滤得结合物，再进行显色反应鉴别。这种情况适合于那些与二抗无法直接结合的菌种。
所述的方法还可以是菌体与标记抗体直接混合进行结合，离心或过滤得结合物，再进行显色反应鉴别，这种情况下适合于那些与标记抗体能直接结合的菌种。
所述的方法既可以通过已知抗体检测未知菌体，也可以通过已知菌体检测未知抗体。通过已知抗体，比如：已知一抗和二抗或者已知标记抗体鉴定菌体的种类。也通过已知菌体来鉴定一抗的种类。
进一步，一抗可以是抗血清、多克隆抗体或单克隆抗体。
进一步，二抗和标记抗体的抗体标记物可以是辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、溶菌酶和苹果酸脱氢酶等。
进一步，菌体与标记抗体二者形成的结合物或菌体与抗体及二抗三者形成的结合物，可以通过离心沉淀并清洗得到，也可以通过滤芯或滤器进行过滤并冲洗得到。
进一步，所涉及的显色底物可以选自3，3′，5，5′-四甲基联苯胺(TMB)和双氧水(H2O2)、3，3′-二氨基偶氮苯(DAB)、邻苯二胺(OPD)、2，2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)中的任一种。
再进一步，显色反应的终止液可以是硫酸、盐酸、叠氮钠等。
本发明可以用于各种细菌的快速检测，包括各种益生菌，如乳酸菌、芽孢菌、酵母菌、丁酸梭菌等各种益生菌株；还包括各种致病菌，如沙门氏菌、大肠杆菌、巴氏杆菌、布氏杆菌、克雷伯氏菌、幽门螺旋杆菌、绿脓杆菌、结核分枝杆菌、炭疽杆菌、坏死杆菌、变形杆菌、志贺氏菌、产气荚膜杆菌、白喉杆菌、脑膜炎球菌、淋球菌、破伤风梭菌、链球菌、金黄色葡萄球菌、放线菌、魏氏梭菌、副嗜血杆菌、丹毒杆菌、李斯特菌、化脓棒状杆菌、钩端螺旋体等各种致病菌株。
免疫共沉淀(Co-Immunoprecipitation，Co-IP)是研究蛋白-蛋白间相互作用的方法，常被用于鉴定特定蛋白复合物中的未知蛋白组分。免疫共沉淀技术的基本策略就是利用抗体-琼脂糖微球与目的蛋白结合共沉淀，清洗掉未结合的杂蛋白，再对沉淀复合物进行变性聚丙烯凝胶电泳(SDS-PAGE)鉴定未知蛋白质。本发明中菌体相当于免疫共沉淀实验当中的微球，这样菌体与相应的抗体相结合后，可以被离心共沉淀下来，再通过酶标抗体与底物发生显色反应，可以快速显色达到鉴定菌体目的，也就是说，与菌体结合的抗体是可以显色的标记抗体，或与菌体一抗结合的二抗是可以显色的标记抗体。所以本发明是将以上Co-IP的共沉淀原理与ELISA的显色原理进行了有机结合，避免了二者的繁琐操作，又降低了成本，成为了一种快速鉴别菌体种类的方法。可以将该方法称为菌体的“免疫沉淀-酶联显色法”。
附图说明
图1屎肠球菌抗体检测的阳性结果图，其中：1为屎肠球菌阳性检测结果；2为屎肠球菌阴性对照
图2屎肠球菌的标记抗体检测的阳性结果图，其中：3为阳性标记抗体的检测结果；4为阴性标记抗体的检测结果
图3肠炎沙门氏菌抗血清检测的阳性结果图，其中：5为肠炎沙门氏菌阳性检测结果；6为肠炎沙门氏菌阴性对照
图4血液中肠炎沙门氏菌抗原检测的阳性结果图，其中：7为肠炎沙门氏菌抗原阴性对照；8为肠炎沙门氏菌抗原阳性检测结果
具体实施方式
实施例1
采用益生菌株屎肠球菌(中国工业微生物菌种保藏管理中心，保藏编号：CICC6049)分别与弗氏佐剂乳化后做为免疫原对家兔进行皮下多点注射免疫，经过3次免疫后，获取兔抗血清，利用辛酸-硫酸铵沉淀法提纯多克隆抗体。接下来按照菌体“免疫沉淀-酶联显色法”操作。将300μL(1mg/ml)多克隆抗体、150μL辣根过氧化物酶标记的羊抗兔IgG(1∶10000稀释)和300μL的屎肠球菌溶液(1×109CFU/ml)三者同时加入离心管中轻轻摇动，室温孵育4-6小时，通过0.22μm滤器进行过滤并反冲洗得到结合物，加入显色底物3，3′，5，5′-四甲基联苯胺(TMB)，立即显示蓝色；将正常兔的阴性抗体作为对照，进行上述相同操作，溶液不显色。结果如图1所示。
实施例2
采用益生菌株屎肠球菌(中国工业微生物菌种保藏管理中心，保藏编号：CICC6049)分别与弗氏佐剂乳化后做为免疫原对家兔进行皮下多点注射免疫，经过3次免疫后，获取兔抗血清，利用辛酸-硫酸铵沉淀法提纯多克隆抗体。将纯化的多克隆抗体采用改良的过碘酸钠法进行辣根过氧化物酶标记；同时制备正常家兔的血清并利用辛酸-硫酸铵沉淀法提纯抗体，也将纯化的抗体采用改良的过碘酸钠法进行辣根过氧化物酶进行标记，将此标记抗体作为阴性对照。接下来按照菌体“免疫沉淀-酶联显色法”操作。将300μL的屎肠球菌溶液(1×109CFU/ml)和150μL辣根过氧化物酶标记兔多克隆抗体(1∶10000稀释)二者同时加入离心管中轻轻摇动，室温孵育4-6小时，通过0.22μm滤器进行过滤并反冲洗得到结合物，加入显色底物TMB，立即显示蓝色；将正常家兔的阴性标记抗体作为对照，进行上述相同操作，溶液不显色。结果如图2所示。
实施例3
将致病菌株肠炎沙门氏菌(中国兽医微生物菌种保藏管理中心，编号CVCC 3377)接种免疫小鼠制备抗血清，同时制备正常小鼠的血清。接下来按照菌体“免疫沉淀-酶联显色法”操作。将100μL抗血清、100μL辣根过氧化物酶标记的羊抗鼠IgG(1∶10000稀释)和100μL的肠炎沙门氏菌溶液(1×109CFU/ml)三者同时加入离心管中轻轻摇动，室温孵育30分钟，此后3000转离心5分钟弃上清，即得菌体沉淀复合物，洗涤3次，加入显色底物TMB，立即显示蓝色；将正常小鼠血清作为对照，进行上述相同操作，溶液不显色。结果如图3所示。
实施例4
将浓度为1×109CFU/ml肠炎沙门氏菌(中国兽医微生物菌种保藏管理中心，编号CVCC3377)给小鼠尾静脉注射，5分钟后小鼠眼眶静脉丛采血0.5ml，EDTA抗凝。接下来利用制备的肠炎沙门氏菌抗血清进行“免疫沉淀-酶联显色法”实验，检测血液中的菌体，正常小鼠血液作为对照。组织细胞裂解液(RIPA)处理抗凝血，裂解血液中细胞，5000转离心5分钟留沉淀，加100μL抗血清、100μL辣根过氧化物酶标记的羊抗鼠IgG(1∶10000稀释)混匀，室温孵育4-6小时，此后5000转离心5分钟弃上清，即得菌体沉淀复合物，用0.01mol/LpH7.4的PBS清洗两次，重悬沉淀用0.22μm滤器进行过滤冲洗，然后反冲洗，加入TMB，立即显示蓝色；正常小鼠血液作为对照，进行上述相同操作，溶液不显色。结果如图4所示。
Claims (8)
1.一种快速检测菌体种类或抗体的方法，将菌体与一抗、二抗混合在一起三者进行结合反应，然后离心或过滤得结合物沉淀，再加入显色底物，进行显色反应鉴别。
2.一种快速检测菌体种类或抗体的方法，其特征在于，所述的菌体与标记抗体二者直接混合进行结合反应，然后离心或过滤得到结合物沉淀，再加入显色底物，进行显色反应鉴别。
3.如权利要求1-2任一项所述的方法，其特征在于，通过已知一抗、二抗检测未知菌体，或通过已知标记抗体检测未知菌体。
4.如权利要求1所述的方法，其特征在于，通过已知菌体检测未知一抗。
5.如权利要求1所述的方法，其特征在于，所述一抗可以是抗血清、多克隆抗体或单克隆抗体。
6.如权利要求1-2任一项所述的方法，其特征在于，二抗和标记抗体的抗体标记物可以是辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、溶菌酶、苹果酸脱氢酶。
7.如权利要求1-2任一项所述的方法，其特征在于，所述的显色底物选自3，3′，5，5′-四甲基联苯胺(TMB)和双氧水(H2O2)、3，3′-二氨基偶氮苯(DAB)、邻苯二胺(OPD)、2，2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)中的任一种。
8.如权利要求1-2任一项所述的方法，其特征在于，显色反应的终止液可以是硫酸、盐酸或叠氮钠。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020090660A1 (en) * | 1999-12-14 | 2002-07-11 | Yi Ching Sui A. | Helicobacter pylori antigens in blood |
| CN101187666A (zh) * | 2007-03-22 | 2008-05-28 | 江苏大学 | 基于elisa法的免疫共沉淀试验方法 |
| US20140315219A1 (en) * | 2013-04-19 | 2014-10-23 | Bret T. Barnhizer | Rapid Enzyme-Linked Immunosorbant Assay for Detection and Identification of Pathogens and Determination of Antimicrobial Susceptibility |
| CN106932575A (zh) * | 2017-03-15 | 2017-07-07 | 北京农学院 | 纳米免疫磁珠技术联合酶联免疫吸附快速检测鼠伤寒沙门氏菌的方法 |
| CN108287235A (zh) * | 2018-02-07 | 2018-07-17 | 常州天地人和生物科技有限公司 | 一种高效、稳定的磁性免疫微球的制备和应用 |
| CN109781995A (zh) * | 2019-01-09 | 2019-05-21 | 吉林特研生物技术有限责任公司 | 一种绿脓杆菌抗体elisa快速检测试剂盒及检测方法 |
-
2019
- 2019-05-30 CN CN201910460420.0A patent/CN110361530A/zh active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020090660A1 (en) * | 1999-12-14 | 2002-07-11 | Yi Ching Sui A. | Helicobacter pylori antigens in blood |
| CN101187666A (zh) * | 2007-03-22 | 2008-05-28 | 江苏大学 | 基于elisa法的免疫共沉淀试验方法 |
| US20140315219A1 (en) * | 2013-04-19 | 2014-10-23 | Bret T. Barnhizer | Rapid Enzyme-Linked Immunosorbant Assay for Detection and Identification of Pathogens and Determination of Antimicrobial Susceptibility |
| CN106932575A (zh) * | 2017-03-15 | 2017-07-07 | 北京农学院 | 纳米免疫磁珠技术联合酶联免疫吸附快速检测鼠伤寒沙门氏菌的方法 |
| CN108287235A (zh) * | 2018-02-07 | 2018-07-17 | 常州天地人和生物科技有限公司 | 一种高效、稳定的磁性免疫微球的制备和应用 |
| CN109781995A (zh) * | 2019-01-09 | 2019-05-21 | 吉林特研生物技术有限责任公司 | 一种绿脓杆菌抗体elisa快速检测试剂盒及检测方法 |
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