| Dengue is an most common arbovirus infection globally caused by dengue virus (DENV). It was transimited among populations by the blood meal of mosquito (major in Aedes Aegypti and Aedes Albopictus). Infection of DENV can induce a broad spectrum of clinical symptoms. Although most of the patients present as a self-limited and flu-like disease, such as dengue fever (DF). However, a minor fraction of patients who suffered from the severe dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS) are threatened by the death unless efficient treatment is received.Presently, dengue is regarded as the most important public health concern due to the high incidence rate and the wide epidemic area, as well as the lack of efficient strategy of prevention and controlling. Over the past50year, the incidence of dengue increased dramatically despite great efforts have been done on the prevention and treatment of the dengue. Dngue brings heavy economical burden to the national healthy system and patient’s families. DENV belongs to Flaviviridae, Flavivirus. It is an enveloped single-strand positive RNA virus. The genome is11kb long, including an open reading frame, coding3structural proteins and7non-structural proteins. These3structural proteins include capsid protein (C), membrane protein (M) and envelop protein (E). These7non-structural proteins are NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5. On the basis of the gene sequence of E protein, DENV can be categoried into four serotypes, DENV-1,-2,-3and-4, with60-70%homology existing among them. The primary infection of dengue can induce life-long immuno-protection against the infected serotype, as well as the transient and mild cross-protection to the other serotypes of DENV. When the serotype of secondary infection is different from the primay infection, the pre-existing non-neutralizing antibodies and sub-neutralizing antibodies can promot the intaking of heterologous DENV into the Fc receptor containing cells, increasing the replications of virus, which is assumed to be antibody dependent enchancement (ADE). ADE is presumed to be a most important factor in the development of severe DHF/DSS.As the efficient dengue vaccine and therapeutics remain unavailable to date, the prevention and controlling of dengue rely heavily on the early, rapid and accurate diagnosis of dengue. Even though robust they are, the three diagnostic golden standards, viral isolation and identification, RT-PCR and sero-diagnosis have their own drawbacks and are not ideal enough for the routine use. For instance, DENV isolation and identification is time-consuming, and presents high false negative rate and requires special laboratory settings and experienced technicians; RT-PCR requires expensive laboratory equipments and experienced technicians. Furthermore, it is hard to standardize. As antibody response usually occurs several days after infection of DENV, and broad cross-reaction within DENV and flavivirus, serodiagnosis of dengue can not be used as early diagnostic tool, and false positive resuts may occurs due to cross-reaction. A simple, convenient, rapid, accurate and cost-effective diagnostic method is still highly desirable for the diagnosis of dengue.NS1is a highly conservative glycoprotein. High level of NS1can be detected in the serum samples on early infection of dengue, of which the level is closely correlated with the severity of disease. Over the last decades, a variety of NS1antigen capture based immunoassays have been established and commercialized. These assays demonstrate a feature of being early, rapid, specific, cheap, easy-to-perform. The serotype-specific NS1antigen capture ELISA based on the elaborately selected NS1mAbs even can be used in serotyping. Thus, immunoassays targeted to NS1antigen play an important role in the diagnosis of dengue. However, the dynamics distribution of NS1protein during different period of infection remain uncharacterized. Full disclosure of kinetics of NS1will be of great help in the analysis the significance and function of NS1antigen detection in the diagnosis of dengue.In our previous studying, a panel of anti-DENV NS1mAbs was prepared uing hybridoma technique, and their immunoactivities were characterized using enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) and western blot (WB). On the basis of optimization and pairing of these NS1mAbs, four DENV serotype-specific NS1antigen capture ELISAs and a group-specific NS1antigen capture ELISA were established with highly sensitive and specific profile. In this study, the kinetics of NS1antigen in the sera of DENV-1patients of priamry infection was analyzed using DENV-1specific antigen capture ELISA, together with the detection of DENV IgM and IgG antibody. The study presented the value of combined assay of antigen and antibody in the diagnosis of dengue.DENV NS1detection not only has great value in the diangosis of dengue, but can be used in the titering of DENV. Furthermore, on the basis of NS1antigen capture ELISA, a micro-neutralizing assay (ELISA-MNT) was established to detect the neutralizing antibody of dengue in this study. Using the ELISA-MNT, the neutralizing antibodies in the sera during period of disease and fellow-up period at interval of more than3years were detected and compared. Coupled with the detection and comparison of other antibodies, e.g. DENV IgG and IgM, anti-DENV EDIII antibody using commercial DENV IgM and IgG antibody capture ELISA, and in-house DENV EDIII antibody capture ELISA, respectively, the kinetics of dengue antibody responses were revealed, which might provided promisng alternatives of measure approaches in the epidemiological survey and the validation of dengue vaccine.Eventhough DENV NS1ELISA is highly sensitive in the diagnosisi of dengue, it is a little complicated in the performance. The colloidal gold assay based rapid diagnosis of dengue is still the goal of clinicians and researchers. However, the sensitivity of the approach is unsatisfactory. In our previous studies, the limit of detection of NS1antigen ELISA was at1:5120dilution of clinical sera, which indicated the high affinity of the used NS1mAbs. However, the high affinity NS1mAbs in ELISA could not be used in collidal gold assay. In this sense, the affinities of these NS1mAbs need to be re-analyzed. In this study, two label-free techniques, the affinities of NS1mAbs were screened using Resonant Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR), and the detailed kinetics information of the interaction between antigens and antibodies were produced for establishement of highly sensitive colloidal gold NS1assay.Thereby, in thist study, the research is divided into three parts:I. Kinetics analysis of NS1antigen, IgM and IgG antibodies in the sera of DENV-1patients of primary infectionDengue NS1antigen detection and seology are the most economical and efficient methods in the diagnosis of dengue. In this study, the kinetics distributions of NS1antigen and IgM and IgG antibodies in the sera of confirmed DENV-1infected patients on the first to27th day after the onset of symptom were detected using in-house DENV-1specific NS1antigen capture ELISA, commercial dengue IgM and IgG antibody capture ELISA, respectively. The results demonstrated that NS1protein could be measured in the sera of87.5%patients on the first day after symptom. The positive detection of NS1ranged from81.8to91.9%during the whole acute phase of clinical course from the first to the seventh day after infection. On the eighth to fourteenth day, i.e. during the early convalescent phase of disease, the detection rate decreased remarkedly. No NS1could be detected in the sera beyond14days, i.e. after the later convalescent phase. In contrast to NS1detection, dengue specific IgM and IgG antibodies appeared in the sera on the third and fifth day, respectively, and the positive detection increased over time. Of both, the detection rate of IgM antibody presented rapider increasing compared to IgG antibodies. IgM peaked to100%detection on the eighth day, whereas, IgG peaked to100%detection on the fifteenth day. The adverse distribution of NS1antigen and IgM antiodies in the sera implied the value of combined detection of both assays. In this study, the combined dection of antigen and antibody increase greatly in the sensitivity of diagnosis, shorten the window phase and extend the time-frame of diagnosis. From the third day when the detection of both assays began to converge, the sensitivity of the combined assay elevated to96.6%to100%, which was significantly higher than that of single NS1antigen or IgM antibody assays. It is indicated that the combined detection of NS1ELISA and IgM ELISA provided a comparatively ideal scheme for the diagnosis of dengue. This scheme basically met the requirements on the ideal diagnosis of dengue with the simple, economical, rapid and efficient profiles.Ⅱ. Determination of antibody response in the consecutive sera of DENV-1confirmed patientsDetection of antibodies plays important role in the diagnosis of dengue, as well as the epidemiological survery and validation of dengue vaccine. However, the dynamics of dengue antibodies throught time is not well-characterized, and the existing methods of antibody detection are not satisfactory enough for the clinical and research works. In this study, two panels of sera samples were detected using commercial dengue IgM and IgG antibody capture ELISA, in-house dengue EDIII IgG antibody capture ELISA and in-house NS1antigen ELISA based micro-neutralizing test (ELISA-MNT). Both panels of sera samples were from the same group of DENV-1patients, which were harvested on the clinical course (the first day to14th day after the onset of symptom) and later convalescent phase (after more than3years of infection). It was obsevered that the dengue IgM and IgG antibodies decayed in the sera over time, whereas the dengue EDⅢ IgG antibodies sustained the stable detection rate over time, which might imply the role of anti-DENV EDⅢ protein in the immuno-pathogenesis. The sensitivity of EDIII IgG ELISA was signifcantly higher than that of dengue IgG ELISA. The ELISA-MNT displayed that the cross-neutralizing activity was higher in the sera harvested during clinical course than that of at later convalescent phase. However, the heterologous neutralizing antibody titers declined over time, or sustained at the low level, whereas the homologous neutralizing antibody titers increased over time, and became the dominant neutralizing antibodies during the later convalescence. Although the EDIII IgG antibodies sustained the stable detection rate over time, they were not associated with the neutralizing antibody titers. Our study revealed the kinetics distribution of antibody responses of dengue infection over time, and provided potentially promising serologies for the diagnosis and epidemiological survery of dengue, although the efficiency need further validation in a larger number of samples.Ⅲ. Evaluation and Comparison of the Relative Binding Affinity of Dengue non-Structural1Antibodies using Label-free Resonant Waveguide Grating and Surface Plasmon Resonance BiosensorsAlthough the NS1antigen capture ELISA established in our laboratory demonstrated to be highly sensitive and specific, the employed NS1monoclonal antibodies (mAbs) were not suitable for the development of colloidal gold assay. The NS1antigen detection based colloidal gold assay is a method of rapid diagnosis of dengue, which can be used to bedside diagnosis and for the rapid screening on field and at custom, and is the priority in the diagnosis of dengue. In this study, two label-free techniques, Resonant Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR) were used to screen and detect the affinity of dengue NS1mAbs. The major objective of this study was to select a panel of efficient antibodies for the establishment of higly sensitive DENV NS1capture based colloidal gold assay. On the basis of immunoactivities of NS1mAbs detected by the labeled immunoassays,80,55,75and64strains of DENV NS1mAbs were designated as the reactive mAbs for DENV-1,-2,-3and-4, respectively. The affinities of these mAbs at saturable status of binded NS1protein to NS1mAbs were screened firstly using the high-throughpput RWG. It was demonstrated that the affinities of these mAbs were ranged from lOnM to1mM in RWG. Then, the high-resolution study on the kinetic interaction of these mAbs was conducted using SPR. SPR can not only provide the affinity (equilibrium dissociation constant, KD), but also the with the association constant (Ka) and dissociation constant (Kd) of the binding of NS1mAbs to NS1protein, as well as the percentage activitiy, which represent the accessibility of antibody to the protein. The affinities of these mAbs were ranged from100nM to1nM in SPR. These mAbs were also the middle to high affinity mAbs (KD=10nM～100nM) in RWG. However, most of these mAbs demonstrated no response in SPR. The association and dissociation constant (Ka and Kd) were ranged from103to105Ms-1and10"7to10-2S-1,respectively in SPR. The percentage activity of DENV-1, DENV-2, DENV-3and DENV-4NS1reactive mAbs was1.6%-47.2%,2.5%-70.3%,1.0%-26.2%fP0.9%-27.2%, respectively. Except that of DENV-4NS1reactive mAbs, the percentage activities of the other three DENV NS1reactive mAbs were significantly higher in high affinity mAbs (10nM) than that of in middle affinity mAbs (100nM) detected by RWG. Collectively, label-free RWG and SPR were potent tools in the identification of affinity between molecules. The high-throughput RWG could be used to screen and rule out a large majority of low affinity mAbs, as well as the mAbs with low percentage activity. SPR could provide high-resolution study for these mAbs on the kinetics interaction between antigen and antibody, and produce detailed information for the subsequent application of NS1mAbs. The resulted data of Ka value from SPR detection was greatly informative for the selection of optimal antibodies in the establishement of NS1detection based colloidal gold assay. However, the efficiency of the colloidal gold assay needed further investigation.Summarization:On the basis of the information resulted from the aforementioned three studies, three major findings and conclusions are attained, which were described as follows:1. On the basis of the in-house DENV-1specific NS1capture ELISA and Panbio dengue IgM and IgG antibody capture ELISAs, the panel of sera samples harvested from DENV-1patients of primary infection on the first day to twenty-senventh day after the onset of symptom were detected in the kinetics distribution of NS1antigen, IgM and IgG antibodies. This study demonstrated value of NS1antigen and IgM antibody detection in the diagnosis of acute and early infection of dengue. The resulted adverse dynamics tendency of NS1antigen and IgM antibody response, indicated that the combined test of NS1antigen and IgM antibody detection could not only greatly increase the diagnostic sensitivity, but also shorten the "window phase"of dengue diagnosis, and extend the digsnostic time-frame.2. The detection of varieties of dengue antibodies in the consecutive sera samples from the same group of DENV-1infected patients harvested at more than3years of interval revealed that the level of dengue IgM and IgG antibodies in the sera samples decayed over time, whereas the level of EDIII IgG antibodies sustained a stable detection rate. The neutralizing antibody titers in the sera samples detected by the in-house ELISA-MNT presented a higher cross-reaction between four serotypes during clinical course than later convalescence. The heterologous cross-reactive neutralizing antibodie declined or sustained at a low level over time, whereas the homologous neutralizing antibody presented an increasing tendency over time, and gradually became the predominant neutralizing antibodies. The current study revealed the kinetic of varieties of dengue antibodies through time after infection of dengue virus, and substantiated that our in-house methods, EDIII IgG antibody capture ELISA and ELISA-MNT might be potential methods in the diagnosis of dengue and epidemiological survey.3. To improve the sensitivity of NS1antigen capture based rapid diagnostic method of dengue, the imrnunochromatographic lateral flow assay (e.g. colloidal gold assay), two label-free techniques, RWG and SPR were used to identify the affinity of NS1mAbs. Of both approaches, RWG is a high-throughput assay, and SPR can be used to high-resolution study on the kinetics interaction between molecules. The study demonstrated that the screening of affinities using RWG at saturable status could not only present the high affinity mAbs, but also the high perecentage activity mAbs. However, the subsequently detection of SPR could produce the association and dissociation constant of these mAbs, of which the association constant provided detailed information for the selection of optimal antibodies for the NS1detection based colloidal gold assay. The combined test of the aforementioned label-free techniques is of a rapid, less-expensive, detailed and accurate scheme in the secreening and selection of favorable mAbs from a large number of mAbs. |
PDF Full Text Request