| Viral hemorrhagic fever (VHF) is a group of a human illnesses caused by dozens of viruses mainly in four distinct of enveloped, single-strand RNA viral families including the Bunyaviridae, Arenaviridae, Filoviridae, and Flaviviridae. As the name implies, the typical clinical symptoms of VHF include fever and bleeding disorders which are often accompanied with headache, muscle joint pain, convulsions and can progress to severe disseminated intravascular coagulation (DIC) and central nervous system damage and even death. Most of the world’s population is now affected by the threat of VHF. So far, VHF discovered in China are mainly hemorrhagic fever with renal syndrome (HFRS), Crimean-Congo hemorrhagic fever (CCHF), dengue hemorrhagic fever (DHF) and severe fever with thrombocytopenia syndrome (SFTS). With the global change of natural environment and rapid development of modern transportation, both the new emerging and the existed hemorrhagic fever viruses (HFVs) showed the trend of rapid spread. Therefore, it is critical to develop laboratory diagnostic system for VHFs control.Currently, diagnosis of VHFs is achieved by RT-PCR, immunofluorescence, ELISA, immunohistochemistry, virus isolation and electron microscopy, and so on. Each method mentioned above cannot meet the requirements of multi-pathogen detection and high-throughput. Serological detection methods are indispensible for viral disease diagnosis including VHF and it needs to screen dozens of viruses that caused VHF and make diagnosis in a short time. Luminex xMAP technology, also known as suspension array or liquid chip, is designed to perform a wide variety of bioassays including multiplexing of up to100specific targets. It combines advanced fluidics, optics, and digital signal processing with proprietary microsphere technology and can be used for high-throughput analysis in a short time, and requires minimal sample volume.In this study, xMAP technology was used as a platform to establish a rapid, sensitive and high-throughput laboratory serological detection method for VHF specific antibodies in order to achieve the purpose of rapid screening and diagnosis for new emerging hemorrhagic fever viruses. Our work can be divided into three parts:1. Expression and purification of29HFVs recombinant proteins.The main purpose of this part was to provide sufficient amounts of high-quality recombinant antigens for establishing VHF multi-serological detection method.29recombinant proteins of HFVs were expressed and then purified using two methods, one was metal ion affinity chromatography or cut specific band of target proteins from gel after SDS-PAGE, and the proteins were eluted from the band of gel which were used for rabbits’immunization to obtain polyclonal sera. SFTSV-NP and HLV-NP were produced from both prokaryotic and eukaryotic systems. The former was used for immunization and the latter for detection. The purity for all29HFVs proteins was above90%as assessed by gel gray scan analysis, meeting the standards for both immunization and detection. In addition, the optimization for each antigen-coating amounts of ELISA detection and evaluation of the corresponding polyclonal rabbit serum were also achieved.2. Establishment and quality control of Luminex-based immunoassay.The purpose of this part is to establish multiplexed serological detection method for VHF. Serological cross-reactivity among genus Phlebovirus was firstly evaluated by ELISA and Western blot analysis, which provided basis for the study of probable existence of cross reactions in the process of the establishment, evaluation and application of Luminex-based immunoassay. Then the coupling effect of each protein to a coded bead set was assessed using corresponding rabbit immune serum and IgG antibody titers were investigated, which could reach to1:4096000for almost all sera. Besides, we also verified that the detection efficiency was not affected obviously after the fluorescent microspheres coupling with antigens stored at4℃for one year and the inter-assay for IgM and IgG detection has no statistical difference. All these work provided foundation for applying this method to practical diagnosis.3. Screening and diagnosis of VHF clinical samples using Luminex-based immunoassayThis part aims to evaluate the clinical diagnostic effect of this newly established method. Cut-off values of10-plexed Luminex-based immunoassay for IgG detection were firstly determined. Numbers of convalescent sera of confirmed HFRS and SFTS patients were selected for IgG detection and sensitivity and specificity were both calculated. Then a number of clinical samples with unknown pathogen infection were detected using this10-plexed immunoassay and several HFRS cases and one DHF case were diagnosed. Based on the above study, cut-off values of29-plexed Luminex-based immunoassay for both IgM and IgG detections were determined and method for removing serum rheumatoid factor was also established. Numbers of sera in both acute and convalescent phase of HFRS and SFTS patients were detected for IgM and IgG antibodies and sensitivity and specificity were determined. A number of clinical samples with unknown pathogen infection were then detected using this29-plexed immunoassay and several VHF positive sera were picked out.In summary, a rapid, sensitive and high-throughput Luminex-based serological detection method for VHF specific antibodies were establish and evaluated, which played an important role in the clinical screening and diagnosis of serum samples and provided a potentially reliable diagnostic tool for serological detection of VHF. |
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