Multiplexed Serological Diagnosis For Viral Hemorrhagic Fever By Suspension Array

Viral hemorrhagic fever is caused by viruses and with fever and bleeding as the main symptoms. All the VHFVs are enveloped and single-stranded RNA virus, including four different viral families, namely, Bunyaviridae, Arenaviridae, Filoviridae, and Flaviviridae. Most of the world population is affected by the threat of VHF. So far, epidemic VHF in China mainly includes hemonrragic fever with renal syndrome (HFRS), Crimean-congo hemorrhagic fever, dengue hemorrhagic fever, severe fever with thrombocytopenia syndrome (SFTS) and so on.Currently, the laboratory diagnostic methods for viral hemorrhagic fever include: ELISA, PCR, IFA, immunohistochemistry, virus isolation, etc. However, the drawback of the laboratory diagnostic methods above is mainly that each response can only test one indicator of the specimens. Although the multiple PCR can simultaneously detect more than one pathgen in one reaction, the quantity of pathogen is limited and this method cann't meet the requirement of high throughput. It's also time consuming, high cost and using of large amount of sample.xMAPTM technology, the multi-functional and multi-analysis synchronization analytical technology, also known as suspension array or liquid chip. It has integrated encoding fluorescent microspheres, laser technology, micro-fluid technology, fast signal processing and data analysis system and provided a relatively high-throughput molecular detection technology platform.In this study, xMAPTm suspension array technology is used in serological diagnosis for viral hemorrhagic fever, to establish a rapid, sensitive, specific and high-throughput method for laboratory diagnosis via detection of specific antibodies of VHF. This research is mainly from the following three parts:1. Expression and purification of the recombinant nucleoprotein antigens of the hemorrhagic fever virusesNine the hemorrhagic fever viral nucleoproteins have been cloned and purified in E coli. The purity of these proteins are all above 90% which were identified by SDS-PAGE. Based on the box titration, it has determined the most appropriate amount of the recombinant nucleoprotein antigens used in detection of the corresponding rabbit sera and the titer of the IgG antibodies in rabbit sera.2. Quality control for the multiplexed suspension array detectionIt has coupled each reconbinant protein antigens of 10 hemorrhagic fever viruses with fluorescent microspheres in 4μg,20μg, 100μg respectively to detect the rabbit sera and then determined the optimal amount of antigen coupling by comparing the results, the rabbit sera were also detected by indirect ELISA. Studied the correlation of the two methods, it has proved that they were highly correlated in a certain range of serum dilution. The coupled microspheres with ten kinds of hemorrhagic fever virus recombinant proteins respectively were mixed to detect each rabbit sera form immunized rabbit by nine of ten antegenis above via quantitative detection of multiplexed fluorescent microspheres. The results of monoplexed and multiplexed were compared to analyse the cross reaction between the different antigens and antibodies. The cross reaction that the protein antigens of different genera hemorrhagic fever virus were used in detection of serological is not obvious. Simultaneous detected the rabbit sera which were removal of E.coli antibodies by affinity chromatography. The resluts prove that E. coli antibodies in serum have limited impact on the data, and the affect is relation with the purity of antigens. 3. Detection of clinical serum by the multiplexed suspension array methodThe fluorescent microspheres, coupled with the recombinant proteins of 10 hemorrhagic fever viruses, were mixed and detected in 5 sera of HFRS patients,5 sera of SFTS patients and 5 sera of normal human respectively. Then it could calculate the value of cutoff, and make a standard curve by serum dilution and fluorescence intensity. By the value of cutoff, it calculated the detection limit and dynamic range of serum samples, which detected by each kind of antigen-coupled fluorescent microspheres, and assessed the specificity of the method. The results had proved that the suspension array of multiplex quantitative detection technology is sensitive and has wide dynamic range in the detection of clinical serum samples. Meanwhile, this method has high specificity in detection of serum from patient who is infected by different species of VHFs.4-plexed fluorescent microspheres (HTN, SEO, SFTS, Dengue) assay was used in detection of IgG antibody of 62 sera of HFRS patients,43 sera of SFTS patients,72 sera of dengue fever patients and 79 nomal sera and IgM antibody of 62 sera of HFRS patients to drawn the ROC curve for selection of the optimal cutoff value and the sensitivity and specificity respectively. It has demonstrated that suspension array of multiplex quantitative detection technology has high sensitivity and specificity. At the same time, after comparing the suspension array method with the immuno chromatography test and enzyme-linked immunosorbent assay respectively, it proved that in qualitation, the suspension array method has higher sensitivity and specificity than immuno chromatography test, and almost same with ELISA.