家蚕成品卵微孢子虫McAb-ELISA检测方法的研究
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摘要
家蚕微粒子病(Pebrine disease)是由家蚕微孢子虫(Nosema bombycis简称Nb)寄生而引起家蚕疾病,是一种分布很广的传染性蚕病,由于其特殊的经卵传染方式,对蚕业生产造成严重威胁,也是蚕种生产上唯一的检疫对象。目前生产上实行母蛾检验制度,淘汰有毒母蛾所产蚕卵,以供应“无毒”蚕种,从而保障了蚕业生产的安全。而成品卵检验检疫作为“补正检查”的方式,近几年也得到广泛关注和作为农业部标准的备检项目,现行生产中的母蛾检验和成品卵检验主要用光学显微镜进行的。随着科学技术的进步和生产实际需要,应用现代分子生物学技术和免疫学技术进行家蚕微粒子病检测实用技术的开发,也是业内十分关注的问题。口岸系统对进出口蚕种承担着较大的责任,目前采用的仍然是显微镜镜检法,存在一定风险。研究开发一种基于免疫学技术,能用于家蚕产品进出口中家蚕微粒子病检疫的方法是必要的。
抗Nb孢子表面蛋白的多抗识别抗原表位多,但与单抗相比,存在交叉反应和灵敏度低等问题。本试验利用多抗识别抗原表位较多、容易捕获孢子和单抗对Nb孢子特异性较强的特点,通过将酶直接标记于单克隆抗体上,意图获得一种简单实用的双抗体夹心ELISA检测方法。
用纯化的家蚕微孢子虫抗兔多抗包被,作为捕获抗原,加入检测样品,然后用辣根过氧化物酶标记的单克隆抗体作为检测抗原,建立双抗体夹心ELISA检测方法,并对各步实验条件进行优化。
研究表明,用液氮冻融加研磨的方法对微孢子虫进行处理后,包被效果显著提高。制备的单抗经western blotting验证,识别的是孢子的表面抗原。多抗最适包被浓度为10μg/ml,最适包被条件为4℃过夜,酶标二抗工作浓度为1:2 000,待检样品和酶标二抗的反应时间分别为37℃1.5h和1h,底物于37℃显色15min。
用所建立的双抗体夹心ELISA检测方法进行了灵敏度检测,结果显示:对纯化孢子的检测量为3.125×105 spores/ml,对“添毒”蚕卵的检测量为5×106 spores/ml。可以用于实际生产中的检测,灵敏度有待于进一步研究加以提高。
Pebrine disease which is caused by Nosema bombycis is the very popular infectious diseases in silkworm and is very wide distributed.Nb is a eukaryotic obligate intracellular parasites.It caused the pebrine disease by parasitized in silkworm bodies and is a destructive disease because of vertical transmission.They cause severe losses to silk farms. Up to now, the detection of microsporidia relied mainly on microscopy mother moth in sericulture,in order to supply non-Nb silkworm eggs. The finished egg quarantine as a "correction check" approach has been wide concern in recent years,and used as the seized items standards by the Ministry of agriculture.The researchers also concerned about the Nb detection by use modern molecular biology and immunology techniques.The port system have the greater responsib- ility to import and export silkworm eggs,there are also certain risk.So, it is essential to study a immunology technology whitch could be used to import and export quarantine in the silkworm products.
The polyclonal antibodies can identify more epitope surface proteins. However, compared with the monoclonal antibody,pAb have more cross-reaction and sensitivity is low.Our study was based on pAb can identify more epitope surface proteins, could capture spores more easy,and monoclonal antibody have more specific characteristics. Through direct marker enzyme in the monoclonal antibody,Intent to obtain a simple and useful antibody-sandwich ELISA detection method. and grinding method of processing Microsporidia Bao was significantly improved results The and antibody,Multi-epitope identification number.
The purified rabbit polyclone antibody of Nosema bombycis was used to be coated on the 96-wellplate, used as capturing antibody,after test samples were added, then Horseradish peroxidase conjugated monoclonal antibodies was added as detecting antibody, and to establish a double-antibody sandwich ELISA assay for the detect,each of steps were optimized.
The study shown that the coating contrast shows that the Nb treated by liquid nitrogen freezing and thawing nitrogen is more easy to coat than the normal Nb.Preparation of the monoclonal antibody verified by western blotting recognition of the spores is the surface antigen.The optimal concentration of recombinant polyclonal antibody for coating of plate was 10 ng/mL, the optimal coating condition for ELISA was 4℃overnight, the work concentration of HRP-labeled monoclonal antibody was 1:2000, the sample for detecting and HRP-labeled monoclonal antibody should be incubated at 37℃for 1.5 h and 1 h respectively, the substrate for ELISA was incubated at 37℃for 15 min before terminated with the stopping solution.
The sensitivity was detected bu use the method condition have established, it was shown that the detected amounts of purified Nb was 3.125×105 spores/mL,and detected amounts of the simulated silkworm rggs infected with Nb was 5×106 spores/mL.The detected amounts need to elevate by further shudies.
抗Nb孢子表面蛋白的多抗识别抗原表位多,但与单抗相比,存在交叉反应和灵敏度低等问题。本试验利用多抗识别抗原表位较多、容易捕获孢子和单抗对Nb孢子特异性较强的特点,通过将酶直接标记于单克隆抗体上,意图获得一种简单实用的双抗体夹心ELISA检测方法。
用纯化的家蚕微孢子虫抗兔多抗包被,作为捕获抗原,加入检测样品,然后用辣根过氧化物酶标记的单克隆抗体作为检测抗原,建立双抗体夹心ELISA检测方法,并对各步实验条件进行优化。
研究表明,用液氮冻融加研磨的方法对微孢子虫进行处理后,包被效果显著提高。制备的单抗经western blotting验证,识别的是孢子的表面抗原。多抗最适包被浓度为10μg/ml,最适包被条件为4℃过夜,酶标二抗工作浓度为1:2 000,待检样品和酶标二抗的反应时间分别为37℃1.5h和1h,底物于37℃显色15min。
用所建立的双抗体夹心ELISA检测方法进行了灵敏度检测,结果显示:对纯化孢子的检测量为3.125×105 spores/ml,对“添毒”蚕卵的检测量为5×106 spores/ml。可以用于实际生产中的检测,灵敏度有待于进一步研究加以提高。
Pebrine disease which is caused by Nosema bombycis is the very popular infectious diseases in silkworm and is very wide distributed.Nb is a eukaryotic obligate intracellular parasites.It caused the pebrine disease by parasitized in silkworm bodies and is a destructive disease because of vertical transmission.They cause severe losses to silk farms. Up to now, the detection of microsporidia relied mainly on microscopy mother moth in sericulture,in order to supply non-Nb silkworm eggs. The finished egg quarantine as a "correction check" approach has been wide concern in recent years,and used as the seized items standards by the Ministry of agriculture.The researchers also concerned about the Nb detection by use modern molecular biology and immunology techniques.The port system have the greater responsib- ility to import and export silkworm eggs,there are also certain risk.So, it is essential to study a immunology technology whitch could be used to import and export quarantine in the silkworm products.
The polyclonal antibodies can identify more epitope surface proteins. However, compared with the monoclonal antibody,pAb have more cross-reaction and sensitivity is low.Our study was based on pAb can identify more epitope surface proteins, could capture spores more easy,and monoclonal antibody have more specific characteristics. Through direct marker enzyme in the monoclonal antibody,Intent to obtain a simple and useful antibody-sandwich ELISA detection method. and grinding method of processing Microsporidia Bao was significantly improved results The and antibody,Multi-epitope identification number.
The purified rabbit polyclone antibody of Nosema bombycis was used to be coated on the 96-wellplate, used as capturing antibody,after test samples were added, then Horseradish peroxidase conjugated monoclonal antibodies was added as detecting antibody, and to establish a double-antibody sandwich ELISA assay for the detect,each of steps were optimized.
The study shown that the coating contrast shows that the Nb treated by liquid nitrogen freezing and thawing nitrogen is more easy to coat than the normal Nb.Preparation of the monoclonal antibody verified by western blotting recognition of the spores is the surface antigen.The optimal concentration of recombinant polyclonal antibody for coating of plate was 10 ng/mL, the optimal coating condition for ELISA was 4℃overnight, the work concentration of HRP-labeled monoclonal antibody was 1:2000, the sample for detecting and HRP-labeled monoclonal antibody should be incubated at 37℃for 1.5 h and 1 h respectively, the substrate for ELISA was incubated at 37℃for 15 min before terminated with the stopping solution.
The sensitivity was detected bu use the method condition have established, it was shown that the detected amounts of purified Nb was 3.125×105 spores/mL,and detected amounts of the simulated silkworm rggs infected with Nb was 5×106 spores/mL.The detected amounts need to elevate by further shudies.
引文
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