抗EV71多克隆抗体偶联的靶向磁性纳米颗粒对病毒的特异性富集

Specific Enrichment of Viral Antigen with EV71 Polyclonal Antibody-coupled Magnetic Particles

手足口病是幼儿和儿童常见的一种传染病,严重时会引起患者死亡。EV71病毒（人肠道病毒71型）是引起手足口病的一种主要病毒。为寻求一条快速诊断EV71病毒感染的新方法,将兔抗EV71多克隆抗体,通过戊二醛交联法偶联在氨基硅烷修饰的磁性纳米颗粒表面,获得抗EV71多克隆抗体偶联的靶向磁性纳米颗粒用于EV71病毒的检测。通过酶联免疫、免疫荧光方法证实了抗体耦合在磁性纳米颗粒表面,并通过BCA法测得其耦合的效率为94.1%。采用抗体偶联的磁性纳米颗粒对EV71病毒液中的病毒抗原进行吸附,通过ELISA法检测上清液中病毒含量的变化,并对磁性纳米颗粒表面吸附的病毒进行免疫荧光和核酸PCR检测,证明了抗体偶联的磁性纳米颗粒可以与病毒抗原特异性结合。由于该纳米颗粒同时具有抗体的靶向性和磁性颗粒的磁响应性,对病毒抗原有较好的特异性吸附能力,可以用于低浓度大样本的EV71病毒抗原的富集检测。利用抗体偶联的靶向磁性纳米颗粒,同时具有可在病毒感染的细胞周围特异性富集和磁颗粒可在交变磁场下感应升温的双重功能,将其作为磁感应热疗的靶向介质,有望研制出病毒感染性疾病磁感应热疗的靶向介质,为靶向热疗病毒感染性疾病提供新的尝试。

Hand, Foot, and Mouth Disease （HFMD） is a common viral illness of infants and children. The disease causes fever and blisterlike eruptions in the mouth and/or a skin rash. Enterovirus 71 （EV71） is known as one of the major causative agents for HFMD, and is sometimes associated with severe central nervous system diseases. In this paper, a novel method for specific enrichment of viral antigen by EV71 polyclonal antibody-coupled magnetic particles is developed. Targeted magnetic nano-particles for specific recognition and enrichment of EV71 antigen were prepared by modifying the polyclonal antibody of EV71 onto the surface of amino-silane coated Magnetic Nanoparticles （MNPs） through glutaraldehyde cross-linking. Antiserum of EV71 was generated by immunization of rabbits with purified EV71 antigen. Next, the serum was precipitated by saturated ammonium sulfate and then was ligated on the surface of Amino-silane-modified magnetic particles through glutaraldehyde cross-linking. Thus, the targeted magnetic medium was prepared for specific enrichment of EV71. Both ELISA and immuno-fluorescence technique were applied to confirm the successful coupling of EV71 polyclonal antibody onto the surface of MNPs, with the coupling efficacy as high as 94.1%. Specific enrichment of the EV71 antigen by the above-mentioned targeted MNPs was studied by immunofluorescence and PCR. Except for the specific enrichment of the virus antigen, with the inductive heating property under Alternative Magnetic Field （AMF）, the antibody coupled MNPs can be further applied as the agent of the Magnetic Mediated Hyperthermia （MMH） for the targeted treatment of infective diseases.