Development and evaluation of an enzyme-linked immunosorbent assay for quantifying antibodies to Japanese encephalitis virus nonstructural 1 protein to detect subclinical infections in vaccinated horses

Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as an alternative to immunostaining. By this method, NS1 antigens contained in culture fluids from cells stably transfected with the NS1 and NS2A genes were captured by a rabbit anti-NS1 polyclonal antibody. Three nanograms per well of NS1 antigen, corresponding to 1:2 to 1:8 dilutions of the culture fluid, was sufficient for testing. ELISA values were obtained by a single-serum dilution (1:100), which correlated with ELISA titers obtained by an endpoint method. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; P < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted no later than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate that the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations.     

A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2 ng ml⁻¹ NS1. Up to 1 μg ml⁻¹ JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10 ng ml⁻¹ was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10 ng ml⁻¹) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.     

Ⅰ型登革病毒NS1抗原捕获ELISA的建立和初步临床诊断应用

目的以登革病毒特异性非结构蛋白1（NS1）单克隆抗体为基础建立Ⅰ型登革病毒（DEN1）抗原检测的酶联免疫吸附（ELISA）法，并探索从病人早期血清样品中检测DEN1-NS1的可行性。方法利用已制备的抗DEN1-NS1单克隆抗体（单抗），进行多种抗体组合配对优化模式的分析，建立双抗体夹心抗原捕获ELISA，以469份健康人血清样品确定cut off值，检测DEN1感染患者急性期血清样品。结果对多种抗体组合反复筛选，最终确立了最佳的包被单抗和酶标测定单抗，建立了抗体夹心捕获DEN1-NS1抗原的酶联免疫测定方法，能特异检测DEN1，与其他血清型登革病毒不发生交叉反应。检测16例临床确诊DEN1感染病人急性期血清样品，15例呈特异的抗原反应阳性。结论成功建立了DEN1-NS1抗原捕获ELISA并应用于临床血清样品的检测，为登革热的早期实验室诊断提供技术方法。     

Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus

Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection.     

Virus-specific antibody-producing cells in blood and cerebrospinal fluid in acute Japanese encephalitis

During an epidemic of Japanese encephalitis (JE) in northern Thailand, cerebrospinal fluid (CSF) leukocytes and blood leukocytes from 28 patients with suspected JE were tested for spontaneous in vitro synthesis of antibodies to JE virus (JEV). Sixteen patients were subsequently proven to be infected with JEV. Supernatant fluids of three-day cultures of unstimulated peripheral blood mononuclear leukocytes or unstimulated unfractionated CSF leukocytes were tested for JEV IgM and IgG antibodies with isotype-specific "antibody capture" radioimmunoassays. Blood-derived leukocytes from all sixteen JEV-infected patients and CSF-derived leukocytes from four JEV-infected patients synthesized JEV antibodies. Blood-derived and CSF-derived leukocytes from all 12 patients with central nervous system infections caused by agents other than JEV uniformly failed to synthesize JEV antibodies. Virus-specific antibody-producing cells can be detected in the blood and CSF early in the clinical course of acute JE.     

Antibody-capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients

Immunoglobulin M (IgM) antibody titers in paired sera from 19 encephalitis and 44 dengue hemorrhagic fever (DHF) patients in Thailand and 42 Japanese encephalitis (JE) patients in Japan were measured by the antibody capture ELISA and applied to distinguish JE virus infection from dengue virus infection. Titer distribution and the ratio of the titers against JE and dengue antigens led to the following diagnostic criteria. The specimens can be considered as positive with JE when IgM-ELISA titer showed over 200 against JE and 4-fold or more higher than titers against any types of dengue antigens. The specimens can be considered as positive with dengue infection when IgM ELISA titer showed over 200 against one of the 4 types of dengue antigens and 4-fold or more higher than against JE antigen. Based on these criteria, 41 of 42 patients in Japan and 11 of 19 encephalitis patients in Thailand could be diagnosed as having JE virus infection while 2 of 19 encephalitis patients in Thailand and 26 of 44 DHF patients in Thailand could be diagnosed as having dengue virus infections.     

Defining the levels of secreted non-structural protein NS1 after West Nile virus infection in cell culture and mice

Infection with West Nile virus (WNV) causes a febrile illness that can progress to meningitis or encephalitis, primarily in humans that are immunocompromised or elderly. For successful treatment of WNV infection, accurate and timely diagnosis is essential. Previous studies have suggested that the flavivirus non-structural protein NS1, a highly conserved and secreted glycoprotein, is a candidate protein for rapid diagnosis. Herein, we developed a capture enzyme-linked immunosorbent assay (ELISA) to detect WNV NS1 using two anti-NS1 monoclonal antibodies (mAbs) that map to distinct sites on the protein. The capture ELISA efficiently detected as little as 0.5 ng/ml of soluble NS1 and exhibited no cross-reactivity for yellow fever, Dengue, and St. Louis encephalitis virus NS1. The capture ELISA reliably detected NS1 in plasma at day 3 after WNV infection, prior to the development of clinical signs of disease. As the time course of infection continued, the levels of detectable NS1 diminished, presumably because of interference by newly generated anti-NS1 antibodies. Indeed, treatment of plasma with a solution that dissociated NS1 immune complexes extended the window of detection. Overall, the NS1-based capture ELISA is a sensitive readout of infection and could be an important tool for diagnosis or screening small molecule inhibitors of WNV infection.     

Development and evaluation of a rapid IgM capture ELISA (JEV-Chex) for the diagnosis of Japanese encephalitis.

BACKGROUND: The IgM capture ELISA has been the most widely used diagnostic method for Japanese encephalitis. However, the lack of availability of validated commercial kits as well as the short shelf life of the kit reagents has limited the use of this technique to very few centres in Asia. OBJECTIVES: Development and evaluation of a rapid IgM capture ELISA (JEV Chex) in comparison to the conventional IgM capture ELISA. Produce key reagents such as cell culture derived JEV antigen and biotinylated monoclonal antibody which are stable at room temperature. STUDY DESIGN: The conventional IgM capture ELISA was modified to reduce the total assay time and two key reagents used in the assay JEV antigen and biotinylated anti-JEV monoclonal antibodies were rendered stable at room temperature using a special procedure. A multi-centric evaluation of this rapid ELISA was carried out using well characterized stored CSF and serum samples. Long term stability of the key reagents was also assessed over a period of 6 months. RESULTS: The rapid IgM capture ELISA developed by us showed complete concordance with the results obtained using the conventional ELISA at all the three centres where it was evaluated. In addition, the stability studies carried out with the inactivated cell culture antigen and the biotinylated monoclonal antibodies stored at room temperature for a period of 180 days revealed that both these reagents yielded consistent optical density values in the ELISA. CONCLUSIONS: The rapid ELISA format of the IgM capture ELISA (JEV-Chex) developed by us as well as the stability of reagents achieved by us in this study is what renders this rapid IgM capture ELISA very robust and user friendly since reagents can be stored at 4 degrees C by peripheral labs.     

Preparation of Japanese encephalitis virus nonstructural protein NS1 obtained from culture fluid of JEV-infected Vero cells

Summary The Japanese encephalitis virus (JEV) nonstructural protein NS1 was released efficiently into culture fluid of JEV-infected Vero cells. The JEV NS1 protein in the infected culture fluid was found almost as a high-molecular-weight form, probably a dimer form of NS1, and was converted to a monomer by boiling. Large amounts of NS1 protein were accumulated in the infected culture fluid. The NS1 protein, separated from JE virions by centrifugation through sucrose layer, could be obtained in large quantities.     

A reverse passive haemagglutination test for detection of Japanese encephalitis virus antigens in cerebrospinal fluid

A rapid sensitive and specific reverse passive haemagglutination test (RPHA) was developed for the detection of Japanese encephalitis virus (JEV) antigens in human cerebrospinal fluid (CSF). Sheep red cells were sensitized with five monoclonal antibodies (109, 112, 203, 204 and 301) reactive with envelope glycoprotein of JEV. Viral antigens were detected in CSF from 35 of 58 (60%) clinical cases of JE when the five MAb coated cells were used in combination. An IgM capture ELISA detected JEV specific antibodies in CSF among 52 of these 58 cases (90%). While 29 specimens contained both antigen and IgM antibodies, 23 had only IgM antibodies and 6 had only antigen. RPHA proved valuable for detection of viral antigens in CSF samples obtained within 10 days after onset of clinical symptoms. Amongst the five MAbs used, the individual antigen detection rates were 44, 12, 43, 12 and 36%, for MAbs 109, 112, 203, 204 and 301, respectively.     

Comparison of Seven Commercial Antigen and Antibody Enzyme-Linked Immunosorbent Assays for Detection of Acute Dengue Infection

Seven commercial assays were evaluated to determine their suitability for the diagnosis of acute dengue infection: (i) the Panbio dengue virus Pan-E NS1 early enzyme-linked immunosorbent assay (ELISA), second generation (Alere, Australia); (ii) the Panbio dengue virus IgM capture ELISA (Alere, Australia); (iii) the Panbio dengue virus IgG capture ELISA (Alere, Australia); (iv) the Standard Diagnostics dengue virus NS1 antigen ELISA (Standard Diagnostics, South Korea); (v) the Standard Diagnostics dengue virus IgM ELISA (Standard Diagnostics, South Korea); (vi) the Standard Diagnostics dengue virus IgG ELISA (Standard Diagnostics, South Korea); and (vii) the Platelia NS1 antigen ELISA (Bio-Rad, France). Samples from 239 Thai patients confirmed to be dengue virus positive and 98 Sri Lankan patients negative for dengue virus infection were tested. The sensitivities and specificities of the NS1 antigen ELISAs ranged from 45 to 57% and 93 to 100% and those of the IgM antibody ELISAs ranged from 85 to 89% and 88 to 100%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics ELISAs gave the best compromise between sensitivity and specificity (87 and 96%, respectively), as well as providing the best sensitivity for patients presenting at different times after fever onset. The Panbio IgG capture ELISA correctly classified 67% of secondary dengue infection cases. This study provides strong evidence of the value of combining dengue virus antigen- and antibody-based test results in the ELISA format for the diagnosis of acute dengue infection.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

Enzyme-linked immunosorbent assay using recombinant antigens for serodiagnosis of Japanese encephalitis

Recombinant Japanese encephalitis (JE) virus proteins were evaluated as antigens for serodiagnosis of JE using an enzyme-linked immunosorbent assay (ELISA). The premembrane/membrane (prM/M) and envelope (E) proteins of JE virus were expressed in HeLa cells infected with a recombinant vaccinia virus that encodes the JE virus prM and E genes and were released from cells in a particulate form. The particulate antigens were partially purified from culture fluid from the infected cells by precipitation of particles with polyethylene glycol and then dissociated from the particles with 0.1% Triton X-100. This antigen preparation was used to evaluate one preimmune and two postvaccination sera from 20 volunteers given three inoculations of the commercial JE vaccine (Biken vaccine) by a conventional ELISA. The results from this assay correlated with neutralization data. The results of an lgM capture ELISA carried out with the recombinant antigen also correlated with the results of an existing lgM capture ELISA performed with JE virus-infected mouse brain, when tested with 29 serum and 13 cerebrospinal fluid samples from JE patients. These results indicated that recombinant JE virus antigens are useful for ELISA as an antigenically equivalent, highly productive, and safe alternative to authentic JE virus antigens. © 1996 Wiley-Liss, Inc.     

西尼罗病毒与乙脑病毒免疫交叉反应的实验研究

为明确西尼罗病毒(WNV)与乙型脑炎病毒(JEV)的免疫交叉反应,本文分别用WNV全抗原与JE减毒活疫苗免疫小鼠,采用间接免疫荧光试验检测血清中2种病毒IgG抗体水平及其交叉反应情况。结果表明:WNV组在第4次免疫后的14天和35天出现2个高峰,平均效价分别为6088和4305;JEV组第4次免疫后,小鼠血清JEV抗体呈现缓慢上升的趋势。无论是在WNV全抗原免疫小鼠血清中还是在JE减毒活疫苗免疫小鼠血清中,同一血清对WNV抗原和JEV抗原均有反应,且抗体效价差异有显著性。在抗WNV抗体阳性血清中,两者交叉反应相对较强,在抗JEV抗体阳性血清中,两者交叉反应较弱。WNV与JEV存在一定交叉反应,但是否有交叉保护作用则需要中和试验等进一步证实。     

Field trial of a Japanese encephalitis diagnostic kit

Serum and cerebrospinal fluid (CSF) obtained from patients in rural Thailand during an encephalitis epidemic were assayed with a Japanese encephalitis rapid diagnosis kit. Japanese encephalitis was diagnosed by detection of virus-specific IgM (JEV IgM) in CSF (1:10 dilution) or serum (1:100 dilution) with an antibody capture enzyme-linked immunosorbent assay. Specimens were assayed immediately on site at the provincial hospital and scored by visual examination within 4 h. Each specimen was retested carefully later to accurately determine its activity (units) at a single screening dilution; each was tested also at serial dilutions to determine its end-point titer. On-site kit results showed close agreement with subsequent laboratory results for detection and quantitation of JEV IgM and JEV IgG in either serum or CSF. Using the kit on site, admission CSF from 35 (73%) of 48 laboratory-proven JEV-infected patients were scored as definitely positive for JEV IgM, while all 17 CSF specimens from non-JEV infected patients were read as negative (sensitivity 73%, specificity 100%). A rapid and early diagnosis of acute Japanese encephalitis can be accomplished almost anywhere.     

抗人μ链单克隆抗体的制备、鉴定及初步应用

目的:制备高效价的鼠源性抗人μ链单克隆抗体(mAb),并建立可用于感染性疾病早期血清学诊断的ELISA捕捉法。方法:以人IgM全分子免疫BALB/c小鼠,按常规方法进行细胞融合,用间接ELISA法筛选及克隆化,建立可稳定分泌抗μ链mAb的杂交瘤细胞株。mAb的特性(效价、Ig亚类、特异性及相对亲和力)采用ELISA及Westernblot法鉴定。以纯化的mAb包被建立ELISA捕捉法,并用于可疑乙脑患者标本中特异性IgM抗体的检测。结果:筛选到1株可稳定分泌抗人μ链mAb的细胞株2E5。mAb腹水的ELISA效价为1×10-6,Ig亚类(型)为IgG1(κ),相对亲和力为1×10-5。Westernblot结果显示mAb2E5仅与IgM的μ链结合,Mr为75000。以辛酸硫酸铵法纯化的mAb2E5包被,建立了ELISA捕捉法,用于30份乙脑患者血清IgM的检测,敏感性及特异性良好。结论:成功地制备1株抗人μ链mAb2E5,建立了可用于感染性疾病早期血清学诊断的ELISA捕捉法。     

Rapid Diagnosis of Japanese Encephalitis by Using an Immunoglobulin M Dot Enzyme Immunoassay

Japanese encephalitis (JE) occurs in rural settings in southern and eastern Asia, where diagnostic facilities are limited. For the diagnosis of JE virus (JEV) infection, we developed a nitrocellulose membrane-based immunoglobulin M (IgM) capture dot enzyme immunoassay (MAC DOT) that is rapid, simple to use, requires no specialized equipment, and can distinguish JEV from dengue infection. In a prospective field study in southern Vietnam, 155 cerebrospinal fluid (CSF) and 341 serum samples were collected from 111 children and 83 adults with suspected encephalitis. The JEV MAC DOT, performed on site, was scored visually from negative to strongly positive by two observers, and the results were compared subsequently with those of the standard IgM capture enzyme-linked immunosorbent assay. For the 179 patients with adequate specimens, the MAC DOT correctly identified 59 of 60 JEV-positive patients and 118 of 119 JEV-negative patients (sensitivity [95% confidence intervals], 98.3% [92.1 to 99.9%]; specificity, 99.2% [95.9 to 100.0%]; positive predictive value, 0.98; negative predictive value, 0.99). The MAC DOT also correctly identified three patients with dengue encephalopathy. Admission specimens were positive for 73% of JE patients. Interobserver agreement for MAC DOT diagnosis was excellent (kappa = 0.94). The JEV MAC DOT is a simple and reliable rapid diagnostic test for JE in rural hospitals.