登革病毒初次感染患者循环登革病毒NS1抗原和抗体的动力学分析以及非标记技术对NS1单克隆抗体亲和力的测定
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摘要
登革热是一种由登革病毒(Dengue Virus, DENV)引起的虫媒传染病,通过蚊子(以埃及伊蚊和白纹伊蚊为主)的叮咬在人群中传播。人类感染DENV后将产生从轻到重的很大范围的临床症候群。尽管绝大多登革热患者显示为种类似流感样的自限性疾病,如登革热(Dengue Fever, DF),然而患者的病情有可能发展到重症登革热,如登革出血热(Dengue Hemorrhage Fever, DHF)或登革休克综合征(Dengue Shock Syndrome, DSS)。在未能得到及时、有效治疗的情况下,重症登革热将对患者的生命造成巨大的威胁。
目前,登革热被认为是世界上最为关注的公共卫生问题,其原因主要在于该病的发病率高,流行范围广,并且缺乏行之有效的防治方法。近50年来,尽管全世界在登革热的防治方面做了很多的努力,但是登革热的发病率还是呈现出急剧上升的趋势,给流行国家的卫生防控体系以及患者的家庭带来了沉重的经济负担。
DENV属于黄病毒科,黄病毒属,是一种有包膜的单股正链的RNA病毒,基因组大约11kb长,包含一个开放性的阅读框,编码3个结构蛋白和7个非结构蛋白。三种结构蛋白分别是衣壳蛋白(capsid protein, C),膜蛋白(membrane protein, M)和包膜蛋白(envelope protein, E)。7种非结构蛋白依次为NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5。根据不同的E蛋白基因序列,DENV可被划分为四种血清型,DENV-1～-4。各种DENV的血清型之间存在60-70%的同源序列。DENV的初次感染可以产生针对感染血清型的终生免疫保护反应,但对其它三型DENV仅有短暂而微弱的交叉免疫保护作用。当二次感染的血清型不同于初次感染的血清型时,患者体内存在的非中和交叉反应性抗体或亚中和浓度的中和抗体可促进DENV进入Fc受体容受细胞,增加病毒的复制,容易导致严重的DHF/DSS的发生。
迄今为止,登革热的防治仍缺乏有效的疫苗和抗病毒治疗方法,由此登革热的防治在很大程度上依赖于对登革热进行早期、快速、精确的诊断。目前登革热的诊断有三个金标准,登革热病毒的分离与鉴定、RT-PCR以及血清学诊断。尽管这三种方法在登革热的诊断中发挥着不可替代的作用,但是每种方法都存在自身的缺陷,例如登革热病毒分离和鉴定需要的时间长,假阴性率高,对实验室和实验技术人员的要求高；RT-PCR不仅对实验室环境、实验设备和实验人员的要求高,而且设备昂贵,难以推广；另外,登革热感染后,患者往往需要一定的时间才能出现抗体反应,而且登革热与黄病毒的抗体之间存在广泛的交叉,因此血清学方法虽然简便、经济,但是不能进行早期诊断,而且可能因为存在黄病毒之间的交叉反应而出现假阳性的结果。由此,建立一种简单、方便、早期、快速、精确、经济的诊断方法仍是登革热研究中亟待解决的问题。
NS1蛋白是DENV中一种保守的糖蛋白,在感染的早期即可在血清中检测到高浓度的存在,并与登革热疾病的严重程度密切相关。近10余年来,各种形式的以NS1抗原捕获为基础的免疫分析法得以建立并商品化。这些NS1抗原检测方法都具有早期、快速、特异、价廉、易于操作等特点。通过对NS1抗体的精心选择而建立的血清学特异性的NS1抗原抗捕获ELISA甚至具有血清分型的作用,因此,以NS1抗原为靶标的免疫分析法在登革热的诊断中具有重要作用。然而,NS1蛋白在登革热感染的不同时期血清中的分布情况仍不甚明了。对于NS1蛋白动力学的充分揭示有助于分析NS1抗原检测在登革热诊断中的意义和作用。
在本实验室的前期工作中,我们用杂交瘤技术制备了一组抗DENV NS1单抗,用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)、免疫荧光试验(immunofluorescence assay, IFA)和蛋白印迹实验(western blot, WB)对这些单抗的免疫反应性进行了分析,在此基础上,通过对这些单抗的优化配对,建立了高度敏感和特异的四种血清型特异性的NS1抗原捕获ELISA,以及一种DENV群特异性的NS1抗原捕获ELISA。在本研究中,利用DENV-1特异性的NS1抗原捕获ELISA,我们动态分析了NS1抗原在DENV-1初次感染患者不同发病时期血清中的分布情况,结合DENV IgM和IgG抗体的检测,揭示了抗原抗体的联合检测在登革热诊断中的作用。
DENV NS1抗原的检测不仅在登革热的诊断中具有很大的优势,而且,基于NS1抗原检测ELISA还具有DENV病毒滴定的作用。此外,利用NS1抗原检测ELISA,在本研究中,我们还建立了一种DENV抗体检测的微中和试验。结合商品化的DENV IgM和IgG抗体捕获ELISA,本实验室自制的DENVE蛋白Ⅲ区(EDⅢ) IgG抗体捕获ELISA,我们对来自同一组DENV-1初次感染患者在发病期和3年后的随诊期血清中的中和抗体、DENV IgM和IgG抗体,EDⅢIgG抗体进行了检测和比较,揭示了随时间的变化DENV患者体内抗体水平的动态变化情况,并为登革热的流行病学调查以及登革热疫苗有效性的验证提供了可能更为有效的检测手段。
尽管NS1抗原检测的ELISA方法在登革热的诊断中具有很好的敏感性,但这种方法的操作还是略嫌复杂。登革热快速诊断用的胶体金法仍是许多临床医生和研究者的目标,但现有的NS1捕获的胶体金法仍缺乏足够的敏感性。在前期工作中,我们发现NS1抗原捕获ELISA的检测极限可达1：5120的血清稀释度,提示这种方法中采用的单抗具有很高的亲和力,但这些ELISA方法中使用的高亲和力NS1单抗并不适合胶体金法。为开发高度敏感的登革热快速诊断的胶体金法,需要对NS1单抗亲和力进行详细的分析。在本研究中,我们采用了非标记技术,共振波导光栅(Resonant Waveguide Grating, RWG)和表面等离激元(Surface Plasmon Resonance, SPR),对大量NS1单抗的亲和力进行了筛选,从而为NS1检测的胶体金法提供了详细的抗原抗体相互作用的动力学信息。
由此,本研究分为以下三个方面的内容：
第一部分：DENV-1初次感染患者血中NS1抗原和IgM和IgG抗体动态分布分析
登革热NS1抗原检测和血清学分析是登革热诊断中最为经济、有效的方法,在登革热的诊断中具有非常重要的地位。在本研究中,我们用自制的DENV-1特异性的NS1抗原捕获ELISA,结合商品化的IgM和IgG抗体捕获ELISA,对广州2006年DENV-1初次感染的确诊患者在发病当天到发病27天的血清标本中的NS1抗原和IgM、IgG抗体在动态分布规律进行了研究。结果发现,在发病的当天,87.5%的患者血中即可检出阳性的NS1蛋白,在发病的第1到第7天,也就是整个登革热病程的急性期,NS1的阳性检测率在81.8%-91.1%之间的高水平检测率范围内波动。而到了发病的第8到14天,也就是疾病的恢复早期,这种NS1检测的敏感性呈现出明显的下降,到发病的第14天后,也就是疾病的恢复晚期,NS1无法测到。与NS1抗原分布不同,IgM和IgG抗体分别在发病的第3和第5天才开始在血中显现,并出现检出率的逐渐升高。其中IgM抗体较IgG抗体不仅出现更早,而且上升的速度更快,在发病的第8天,IgM的检测达到检测高峰的100%,而IgG抗体则在发病的第15天才达到检测的高峰(100%)。NS1抗原和IgM抗体在血中的分布呈现一种相反的互补规律,当用两者方法进行联合检测时,可大大提高登革热诊断的敏感性、缩短检测的窗口期、延长检测的时限。表现为在两者的检出开始出现交叉的第3天一直到恢复期,联合诊断的阳性率可高达96.6-100%,明显高于单独的NS1抗原或IgM抗体检测的敏感性。研究表明,登革热NS1抗原和IgM、IgG抗体的联合检测为不同感染时期的初次感染患者提供了个较为理想的登革热诊断方案,这种方案基本上可以满足对登革热进行简单、经济、快速、有效的诊断要求。
第二部分：1型登革病毒初次感染患者急性期和康复期血清学动态分析
登革热患者血清中抗体的检测不仅对登革热的诊断,而且在登革热流行病学调查以及疫苗有效性的验证等方面具有重要作用,但登革热感染后,各种抗体随时间的动态变化情况尚未得到充分阐明,而且现有的登革热抗体检测方法仍无法充分满足临床和研究的需要。在本研究中,利用DENV NS1抗原捕获ELISA,我们建立了一种DENV中和抗体检测的微中和试验,结合商品化的Panbio IgM和IgG抗体捕获ELISA,以及自制的抗登革热EDⅢ IgG抗体捕获ELISA对DENV-1初次感染患者在发病期以及3年余后随诊期的血清标本进行了DENV相关抗体的检测。研究发现,登革热特异性的IgM和IgG抗体的检出率随时间的推移而发生下降,但抗EDⅢ IgG抗体的检出率随时间的推移在体内继续保持着稳定的检出率。在随诊期,抗EDⅢ IgG抗体捕获ELISA对血清标本检测的敏感性显著高于Panbio IgG抗体捕获ELISA。这一结果不仅提示了EDⅢIgG ELISA是一种更为敏感的抗体检测方法,而且EDⅢ IgG抗体在体内的稳定检出率还提示了EDⅢ蛋白在登革热的免疫发病机制中可能存在重要作用。而微中和实验显示,发病期血清的中和抗体在四型DENV中具有很强的交叉中和活性,但随时间的推移,异型中和抗体滴度出现下降,或者是持续维持着一个低水平,而同型中和抗体则随时间推移出现滴度的上升,到随诊期,血清中同型中和抗体成为了最具优势的中和抗体。但本研究未能显示EDⅢ IgG抗体与中和抗体之间存在统计学上的相关性。本研究不仅揭示了登革热感染后,各种抗体随时间的变化而发生动态变化的规律,而且表明,本研究室自制的抗登革热EDⅢIgG抗体捕获ELISA,以及基于NS1抗原ELISA的微中和实验可能是登革热诊断和流行病学调查的有效的检测工具,尽管其有效性仍需要通过对更大样本的进一步检测而得证实。
第三部分：共振波导光栅和表面等离激元技术对登革热NS1单抗与NS1蛋白亲和力的鉴定与比较
尽管我们建立的DENV NS1ELISA具有较好的敏感性和特异性,但是这些NS1ELISA中所采用的抗体并不适用于胶体金法。基于DENV NS1抗原检测的胶体金法是一种DENV的快速诊断方法,可以作为床边诊断(bedside diagnosis)以及现场、出入境的快速筛查,是DENV诊断的研究热点。为寻找胶体金法NS1抗原检测法适合的单抗,在本研究中,我们采用两种免标记(non-labeled)技术,共振波导光栅(Resonant Waveguide Grating, RWG)和表面等离激元(Surface Plasmon Resonance, SPR)对DENV NS1单抗与NS1蛋白的亲和性进行了进一步的分析。在前期利用免疫标记技术获得的DENV NS1mAbs的免疫反应性的基础上,我们选出了80株,55株,75株和64株分别针对DENV-1, DENV-2, DENV-3和DENV-4NS1的反应性单抗。先用RWG这种高通量的免标记技术对这些单抗在饱和结合状态下的亲和力进行了初筛。结果显示,这些抗体的亲和力的范围在10nM-1mM之间。随后用SPR对这些抗体与抗原相互作用的动态过程进行了进一步的详细分析。SPR方法的检测不仅可以提供NS1单抗与NS1蛋白的亲和力,也就是平衡解离常数(equilibrium dissociation constant, KD),而且可以进一步提供两者之间的结合常数(association constant, Ka)及解离常数(dissociation constant, Kd),以及代表NS1单抗对NS1蛋白可及性(accessibility)的单抗活性百分数。在本研究中,用SPR得出NS1单抗的亲和力范围位于1nM～100nM之间,这些单抗同时也是RWG方法检测到的高、中亲和力的单抗(KD=10nM-100nM),但绝大部分抗体在SPR中显现为无反应性。SPR检测出的NS1单抗的K。和&则分别波动在103到105Ms-1之间和10-7到10-2S-1之间。而DENV-1, DENV-2, DENV-3和DENV-4NS1反应性的单抗的活性百分数则分别波动在1.6%-47.2%,2.5%-70.3%,1.0%-26.2%和0.9%-27.2%之间。除DENV-4NS1反应性单抗外,在RWG所测得的高亲和力(KD=1OnM)单抗,其在SPR中测得的活性百分数显著高于RWG测得为中等亲和力(KD=1OOnM)的单抗。研究表明,免标记的RWG和SPR技术是检测分子之间相互作用的亲和力的有效工具。当对大量标本进行筛查时,可先用高通量的RWG可以对大量的抗体初筛,排除低亲和力和低活性百分数的标本,然后用SPR对高亲和力和高活性百分数的抗体进行进一步动力学分析。根据SPR的检测结果,一些具有高结合常数的NS1单抗可作为胶体金的NS1抗原检测方法中的检测和捕获抗体,尽管其有效性仍需要得到进一步的验证。
小结
通过以上三部分的研究结果发现,本研究的具有以下三个创新之处,其中包括：
一、利用我们建立的DENV-1特异性NS1抗原捕获ELISA和商品化的Panbio IgM和IgG抗体捕获ELISA,观察了DENV-1初次感染确诊患者从发病的第1天到第27天血清中NS1抗原和IgM、IgG抗体水平的动态变化情况。首次揭示了登革初次感染患者DENV NS1抗原和IgM抗体互补的消长规律,提示了NS1抗原检测在急性期以及IgM抗体检测在恢复早期登革热诊断中的作用,而这两种方法的联合检测,不仅可以大大提高登革热诊断的敏感性,而且可以缩短登革热诊断的“窗口期”,延长登革热诊断的时限。
二、在本研究中,通过对同一组患者在发病期和随诊期血清中各种抗体动态分析发现,登革热特异性的IgM和IgG抗体的检出率随时间的推移发生衰退,而抗EDⅢ IgG抗体随时间的推移依然保持着稳定的检出率,提示自制的DENV EDⅢ IgG抗体捕获ELISA有可能是一种有效的登革热血清流行病学调查的工具。而基于NS1抗原检测的微中和实验对系列血清中的中和抗体检测提示,发病期血清的中和抗体在四型DENV中具有更强的交叉中和活性,但异型中和抗体滴度随时间的推移出现下降,或者是一直维持着一个低水平,而同型中和抗体随时间推移则出现上升,并逐渐显示为优势的抗体中和效价。本研究不仅揭示了登革热感染后,各种抗体随时间的变化而发生动态变化情况,而且为登革热的诊断和流行病学调查提供了可能较为有效的登革热感染的血清学检测方法。
三、为建立登革热进行快速诊断的NS1抗原捕获胶体金法,我们采用了两种检测分子之间相互作用的免标记技术,RWG和SPR对DENV NS1单抗与NS1蛋白的亲和力进行的鉴定与比较。其中,RWG是一种高通量的免标记技术,而SPR可对分子之间相互作用的动力学信息进行详细地分析。研究发现,RWG对抗原抗体结合的饱和状态下的亲和力的筛查,不仅可以检测出在SPR中高亲和力的抗体,而且还能检测出高活性百分数的抗体。而SPR的进一步分析提供了抗体的结合常数和解离常数,其中抗体的结合常数将为胶体金法的NS1抗原检测最佳的抗体候选物的选择提供了详细的信息。两种非标记技术的结合,为大量NS1单抗亲和力的检测提供了一个快速、经济、详细、精确的途径。
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 infection
Dengue 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 patients
Detection 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 Biosensors
Although 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.
目前,登革热被认为是世界上最为关注的公共卫生问题,其原因主要在于该病的发病率高,流行范围广,并且缺乏行之有效的防治方法。近50年来,尽管全世界在登革热的防治方面做了很多的努力,但是登革热的发病率还是呈现出急剧上升的趋势,给流行国家的卫生防控体系以及患者的家庭带来了沉重的经济负担。
DENV属于黄病毒科,黄病毒属,是一种有包膜的单股正链的RNA病毒,基因组大约11kb长,包含一个开放性的阅读框,编码3个结构蛋白和7个非结构蛋白。三种结构蛋白分别是衣壳蛋白(capsid protein, C),膜蛋白(membrane protein, M)和包膜蛋白(envelope protein, E)。7种非结构蛋白依次为NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5。根据不同的E蛋白基因序列,DENV可被划分为四种血清型,DENV-1～-4。各种DENV的血清型之间存在60-70%的同源序列。DENV的初次感染可以产生针对感染血清型的终生免疫保护反应,但对其它三型DENV仅有短暂而微弱的交叉免疫保护作用。当二次感染的血清型不同于初次感染的血清型时,患者体内存在的非中和交叉反应性抗体或亚中和浓度的中和抗体可促进DENV进入Fc受体容受细胞,增加病毒的复制,容易导致严重的DHF/DSS的发生。
迄今为止,登革热的防治仍缺乏有效的疫苗和抗病毒治疗方法,由此登革热的防治在很大程度上依赖于对登革热进行早期、快速、精确的诊断。目前登革热的诊断有三个金标准,登革热病毒的分离与鉴定、RT-PCR以及血清学诊断。尽管这三种方法在登革热的诊断中发挥着不可替代的作用,但是每种方法都存在自身的缺陷,例如登革热病毒分离和鉴定需要的时间长,假阴性率高,对实验室和实验技术人员的要求高；RT-PCR不仅对实验室环境、实验设备和实验人员的要求高,而且设备昂贵,难以推广；另外,登革热感染后,患者往往需要一定的时间才能出现抗体反应,而且登革热与黄病毒的抗体之间存在广泛的交叉,因此血清学方法虽然简便、经济,但是不能进行早期诊断,而且可能因为存在黄病毒之间的交叉反应而出现假阳性的结果。由此,建立一种简单、方便、早期、快速、精确、经济的诊断方法仍是登革热研究中亟待解决的问题。
NS1蛋白是DENV中一种保守的糖蛋白,在感染的早期即可在血清中检测到高浓度的存在,并与登革热疾病的严重程度密切相关。近10余年来,各种形式的以NS1抗原捕获为基础的免疫分析法得以建立并商品化。这些NS1抗原检测方法都具有早期、快速、特异、价廉、易于操作等特点。通过对NS1抗体的精心选择而建立的血清学特异性的NS1抗原抗捕获ELISA甚至具有血清分型的作用,因此,以NS1抗原为靶标的免疫分析法在登革热的诊断中具有重要作用。然而,NS1蛋白在登革热感染的不同时期血清中的分布情况仍不甚明了。对于NS1蛋白动力学的充分揭示有助于分析NS1抗原检测在登革热诊断中的意义和作用。
在本实验室的前期工作中,我们用杂交瘤技术制备了一组抗DENV NS1单抗,用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)、免疫荧光试验(immunofluorescence assay, IFA)和蛋白印迹实验(western blot, WB)对这些单抗的免疫反应性进行了分析,在此基础上,通过对这些单抗的优化配对,建立了高度敏感和特异的四种血清型特异性的NS1抗原捕获ELISA,以及一种DENV群特异性的NS1抗原捕获ELISA。在本研究中,利用DENV-1特异性的NS1抗原捕获ELISA,我们动态分析了NS1抗原在DENV-1初次感染患者不同发病时期血清中的分布情况,结合DENV IgM和IgG抗体的检测,揭示了抗原抗体的联合检测在登革热诊断中的作用。
DENV NS1抗原的检测不仅在登革热的诊断中具有很大的优势,而且,基于NS1抗原检测ELISA还具有DENV病毒滴定的作用。此外,利用NS1抗原检测ELISA,在本研究中,我们还建立了一种DENV抗体检测的微中和试验。结合商品化的DENV IgM和IgG抗体捕获ELISA,本实验室自制的DENVE蛋白Ⅲ区(EDⅢ) IgG抗体捕获ELISA,我们对来自同一组DENV-1初次感染患者在发病期和3年后的随诊期血清中的中和抗体、DENV IgM和IgG抗体,EDⅢIgG抗体进行了检测和比较,揭示了随时间的变化DENV患者体内抗体水平的动态变化情况,并为登革热的流行病学调查以及登革热疫苗有效性的验证提供了可能更为有效的检测手段。
尽管NS1抗原检测的ELISA方法在登革热的诊断中具有很好的敏感性,但这种方法的操作还是略嫌复杂。登革热快速诊断用的胶体金法仍是许多临床医生和研究者的目标,但现有的NS1捕获的胶体金法仍缺乏足够的敏感性。在前期工作中,我们发现NS1抗原捕获ELISA的检测极限可达1：5120的血清稀释度,提示这种方法中采用的单抗具有很高的亲和力,但这些ELISA方法中使用的高亲和力NS1单抗并不适合胶体金法。为开发高度敏感的登革热快速诊断的胶体金法,需要对NS1单抗亲和力进行详细的分析。在本研究中,我们采用了非标记技术,共振波导光栅(Resonant Waveguide Grating, RWG)和表面等离激元(Surface Plasmon Resonance, SPR),对大量NS1单抗的亲和力进行了筛选,从而为NS1检测的胶体金法提供了详细的抗原抗体相互作用的动力学信息。
由此,本研究分为以下三个方面的内容：
第一部分：DENV-1初次感染患者血中NS1抗原和IgM和IgG抗体动态分布分析
登革热NS1抗原检测和血清学分析是登革热诊断中最为经济、有效的方法,在登革热的诊断中具有非常重要的地位。在本研究中,我们用自制的DENV-1特异性的NS1抗原捕获ELISA,结合商品化的IgM和IgG抗体捕获ELISA,对广州2006年DENV-1初次感染的确诊患者在发病当天到发病27天的血清标本中的NS1抗原和IgM、IgG抗体在动态分布规律进行了研究。结果发现,在发病的当天,87.5%的患者血中即可检出阳性的NS1蛋白,在发病的第1到第7天,也就是整个登革热病程的急性期,NS1的阳性检测率在81.8%-91.1%之间的高水平检测率范围内波动。而到了发病的第8到14天,也就是疾病的恢复早期,这种NS1检测的敏感性呈现出明显的下降,到发病的第14天后,也就是疾病的恢复晚期,NS1无法测到。与NS1抗原分布不同,IgM和IgG抗体分别在发病的第3和第5天才开始在血中显现,并出现检出率的逐渐升高。其中IgM抗体较IgG抗体不仅出现更早,而且上升的速度更快,在发病的第8天,IgM的检测达到检测高峰的100%,而IgG抗体则在发病的第15天才达到检测的高峰(100%)。NS1抗原和IgM抗体在血中的分布呈现一种相反的互补规律,当用两者方法进行联合检测时,可大大提高登革热诊断的敏感性、缩短检测的窗口期、延长检测的时限。表现为在两者的检出开始出现交叉的第3天一直到恢复期,联合诊断的阳性率可高达96.6-100%,明显高于单独的NS1抗原或IgM抗体检测的敏感性。研究表明,登革热NS1抗原和IgM、IgG抗体的联合检测为不同感染时期的初次感染患者提供了个较为理想的登革热诊断方案,这种方案基本上可以满足对登革热进行简单、经济、快速、有效的诊断要求。
第二部分：1型登革病毒初次感染患者急性期和康复期血清学动态分析
登革热患者血清中抗体的检测不仅对登革热的诊断,而且在登革热流行病学调查以及疫苗有效性的验证等方面具有重要作用,但登革热感染后,各种抗体随时间的动态变化情况尚未得到充分阐明,而且现有的登革热抗体检测方法仍无法充分满足临床和研究的需要。在本研究中,利用DENV NS1抗原捕获ELISA,我们建立了一种DENV中和抗体检测的微中和试验,结合商品化的Panbio IgM和IgG抗体捕获ELISA,以及自制的抗登革热EDⅢ IgG抗体捕获ELISA对DENV-1初次感染患者在发病期以及3年余后随诊期的血清标本进行了DENV相关抗体的检测。研究发现,登革热特异性的IgM和IgG抗体的检出率随时间的推移而发生下降,但抗EDⅢ IgG抗体的检出率随时间的推移在体内继续保持着稳定的检出率。在随诊期,抗EDⅢ IgG抗体捕获ELISA对血清标本检测的敏感性显著高于Panbio IgG抗体捕获ELISA。这一结果不仅提示了EDⅢIgG ELISA是一种更为敏感的抗体检测方法,而且EDⅢ IgG抗体在体内的稳定检出率还提示了EDⅢ蛋白在登革热的免疫发病机制中可能存在重要作用。而微中和实验显示,发病期血清的中和抗体在四型DENV中具有很强的交叉中和活性,但随时间的推移,异型中和抗体滴度出现下降,或者是持续维持着一个低水平,而同型中和抗体则随时间推移出现滴度的上升,到随诊期,血清中同型中和抗体成为了最具优势的中和抗体。但本研究未能显示EDⅢ IgG抗体与中和抗体之间存在统计学上的相关性。本研究不仅揭示了登革热感染后,各种抗体随时间的变化而发生动态变化的规律,而且表明,本研究室自制的抗登革热EDⅢIgG抗体捕获ELISA,以及基于NS1抗原ELISA的微中和实验可能是登革热诊断和流行病学调查的有效的检测工具,尽管其有效性仍需要通过对更大样本的进一步检测而得证实。
第三部分：共振波导光栅和表面等离激元技术对登革热NS1单抗与NS1蛋白亲和力的鉴定与比较
尽管我们建立的DENV NS1ELISA具有较好的敏感性和特异性,但是这些NS1ELISA中所采用的抗体并不适用于胶体金法。基于DENV NS1抗原检测的胶体金法是一种DENV的快速诊断方法,可以作为床边诊断(bedside diagnosis)以及现场、出入境的快速筛查,是DENV诊断的研究热点。为寻找胶体金法NS1抗原检测法适合的单抗,在本研究中,我们采用两种免标记(non-labeled)技术,共振波导光栅(Resonant Waveguide Grating, RWG)和表面等离激元(Surface Plasmon Resonance, SPR)对DENV NS1单抗与NS1蛋白的亲和性进行了进一步的分析。在前期利用免疫标记技术获得的DENV NS1mAbs的免疫反应性的基础上,我们选出了80株,55株,75株和64株分别针对DENV-1, DENV-2, DENV-3和DENV-4NS1的反应性单抗。先用RWG这种高通量的免标记技术对这些单抗在饱和结合状态下的亲和力进行了初筛。结果显示,这些抗体的亲和力的范围在10nM-1mM之间。随后用SPR对这些抗体与抗原相互作用的动态过程进行了进一步的详细分析。SPR方法的检测不仅可以提供NS1单抗与NS1蛋白的亲和力,也就是平衡解离常数(equilibrium dissociation constant, KD),而且可以进一步提供两者之间的结合常数(association constant, Ka)及解离常数(dissociation constant, Kd),以及代表NS1单抗对NS1蛋白可及性(accessibility)的单抗活性百分数。在本研究中,用SPR得出NS1单抗的亲和力范围位于1nM～100nM之间,这些单抗同时也是RWG方法检测到的高、中亲和力的单抗(KD=10nM-100nM),但绝大部分抗体在SPR中显现为无反应性。SPR检测出的NS1单抗的K。和&则分别波动在103到105Ms-1之间和10-7到10-2S-1之间。而DENV-1, DENV-2, DENV-3和DENV-4NS1反应性的单抗的活性百分数则分别波动在1.6%-47.2%,2.5%-70.3%,1.0%-26.2%和0.9%-27.2%之间。除DENV-4NS1反应性单抗外,在RWG所测得的高亲和力(KD=1OnM)单抗,其在SPR中测得的活性百分数显著高于RWG测得为中等亲和力(KD=1OOnM)的单抗。研究表明,免标记的RWG和SPR技术是检测分子之间相互作用的亲和力的有效工具。当对大量标本进行筛查时,可先用高通量的RWG可以对大量的抗体初筛,排除低亲和力和低活性百分数的标本,然后用SPR对高亲和力和高活性百分数的抗体进行进一步动力学分析。根据SPR的检测结果,一些具有高结合常数的NS1单抗可作为胶体金的NS1抗原检测方法中的检测和捕获抗体,尽管其有效性仍需要得到进一步的验证。
小结
通过以上三部分的研究结果发现,本研究的具有以下三个创新之处,其中包括：
一、利用我们建立的DENV-1特异性NS1抗原捕获ELISA和商品化的Panbio IgM和IgG抗体捕获ELISA,观察了DENV-1初次感染确诊患者从发病的第1天到第27天血清中NS1抗原和IgM、IgG抗体水平的动态变化情况。首次揭示了登革初次感染患者DENV NS1抗原和IgM抗体互补的消长规律,提示了NS1抗原检测在急性期以及IgM抗体检测在恢复早期登革热诊断中的作用,而这两种方法的联合检测,不仅可以大大提高登革热诊断的敏感性,而且可以缩短登革热诊断的“窗口期”,延长登革热诊断的时限。
二、在本研究中,通过对同一组患者在发病期和随诊期血清中各种抗体动态分析发现,登革热特异性的IgM和IgG抗体的检出率随时间的推移发生衰退,而抗EDⅢ IgG抗体随时间的推移依然保持着稳定的检出率,提示自制的DENV EDⅢ IgG抗体捕获ELISA有可能是一种有效的登革热血清流行病学调查的工具。而基于NS1抗原检测的微中和实验对系列血清中的中和抗体检测提示,发病期血清的中和抗体在四型DENV中具有更强的交叉中和活性,但异型中和抗体滴度随时间的推移出现下降,或者是一直维持着一个低水平,而同型中和抗体随时间推移则出现上升,并逐渐显示为优势的抗体中和效价。本研究不仅揭示了登革热感染后,各种抗体随时间的变化而发生动态变化情况,而且为登革热的诊断和流行病学调查提供了可能较为有效的登革热感染的血清学检测方法。
三、为建立登革热进行快速诊断的NS1抗原捕获胶体金法,我们采用了两种检测分子之间相互作用的免标记技术,RWG和SPR对DENV NS1单抗与NS1蛋白的亲和力进行的鉴定与比较。其中,RWG是一种高通量的免标记技术,而SPR可对分子之间相互作用的动力学信息进行详细地分析。研究发现,RWG对抗原抗体结合的饱和状态下的亲和力的筛查,不仅可以检测出在SPR中高亲和力的抗体,而且还能检测出高活性百分数的抗体。而SPR的进一步分析提供了抗体的结合常数和解离常数,其中抗体的结合常数将为胶体金法的NS1抗原检测最佳的抗体候选物的选择提供了详细的信息。两种非标记技术的结合,为大量NS1单抗亲和力的检测提供了一个快速、经济、详细、精确的途径。
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 infection
Dengue 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 patients
Detection 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 Biosensors
Although 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.
引文
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