Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B

An amino acid consensus sequence for the seven serotypes of foot-and-mouth disease virus (FMDV) nonstructural protein 3B, including all three contiguous repeats, and its use in the development of a pan-serotype diagnostic test for all seven FMDV serotypes are described. The amino acid consensus sequence of the 3B protein was determined from a multiple-sequence alignment of 125 sequences of 3B. The consensus 3B (c3B) protein was expressed as a soluble recombinant fusion protein with maltose-binding protein (MBP) using a bacterial expression system and was affinity purified using amylose resin. The MBP-c3B protein was used as the antigen in the development of a competition enzyme-linked immunosorbent assay (cELISA) for detection of anti-3B antibodies in bovine sera. The comparative diagnostic sensitivity and specificity at 47% inhibition were estimated to be 87.22% and 93.15%, respectively. Reactivity of c3B with bovine sera representing the seven FMDV serotypes demonstrated the pan-serotype diagnostic capability of this bioreagent. The consensus antigen and competition ELISA are described here as candidates for a pan-serotype diagnostic test for FMDV infection.     

Recombinant Hepatitis A Virus Antigen: Improved Production and Utility in Diagnostic Immunoassays

Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted.     

乙型肝炎病毒前S1抗原检测的临床意义

探讨乙型肝炎病毒前s1抗原（Pre-S1）检测在乙型肝炎病毒诊断中的临床意义。采用酶联免疫吸附试验（ELISA）和荧光定量聚合酶链反应技术（fluorescenee quantitative PCR,FQ-PCR）对650份HBV-M不同阳性模式及40份HBV—M全阴性模式血清标本进行乙型肝炎病毒Pre-S1、乙肝五项和HBV—DNA检测,并对三种检测结果进行统计学分析。在650份HBV—M不同阳性模式标本中,在119份大三阳标本中Pre—S1阳性检出率92．4％,HBV-DNA阳性检出率100％,在186份小三阳标本中Pre—SI阳性检出率42．5％,HBV—DNA阳性检出率63．4％,在21例HBsAg（＋）和HBcAb（＋）阳性组中Pres1阳性检出率47．6％,HBV．DNA阳性检出率66．7％;在297例HBsAb（＋）标本中Pre—S1阳性检出率0．4％,HBV-DNA阳性检出率0％,在268例HBV—DNA阳性的标本中Pre-S1阳性检出率79．3％。在40份HBV—M全阴模式中Pre-S1阳性检出率0％,HBV-DNA阳性检出率0％。Pre—S1在大三阳、小三阳及HBV-DNA阳性组阳性检出率明显高于阴性组（P〈0．01）,Pre—S1检测可补充和完善乙肝“两对半”检测的不足,尤其对HBeAg阴性或变异的HBV感染者能更好的反映病毒的复制状态和传染性。     

Comparison of Capture Immunoglobulin M (IgM) and IgG Enzyme-Linked Immunosorbent Assay (ELISA) and Nonstructural Protein NS1 Serotype-Specific IgG ELISA for Differentiation of Primary and Secondary Dengue Virus Infections

We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.     

固相酶联免疫测定法检测NS1抗原在登革病毒感染早期诊断中的应用

目的探讨固相酶联免疫测定（ELISA）法检测NS1抗原在登革病毒感染早期诊断中的应用价值。方法选取登革病毒感染早期患者血清171份,非登革病毒感染发热患者血清11份,正常人血清10份,采用ELISA法检测全部192份血清的登革病毒NS1抗原和IgM抗体;采用逆转录-聚合酶链反应-限制性内切酶酶切片段长度多态性分析（RT-PCR-RFLP）技术对发病5 d内的125份血清进行扩增和鉴定分型;并采用C6/36细胞微量培养法对发病第1、2天的41份血清进行登革病毒分离培养。结果登革病毒感染患者发病2 d内、3～5 d以及6～10 d血清NS1抗原的检出率分别是92.7%（38/41）、83.3%（70/84）、10.9%（5/46）;IgM抗体的检出率分别是2.4%（1/41）、51.2%（43/84）、97.8%（45/46）;非登革病毒感染的发热患者及正常人血清中,有1例疟疾患者血清登革病毒IgM抗体呈阳性,NS1抗原无一例阳性。RT-PCR在登革病毒感染患者发病第1、2天和3～5天的检出率分别是85.4%（35/41）、83.3%（70/84）;登革病毒感染患者发病第1、2天血清的病毒分离培养阳性率分别是80.0%（16/20）、38.1%（8/21）,总分离率58.5%（24/41）;RT-PCR-RFLP分型鉴定技术及间接免疫荧光法（IFA）均证实2006年广州流行株为登革Ⅰ型病毒。结论ELISA法检测登革病毒NS1抗原操作技术成熟,且具有敏感性高、特异性好的特点,对登革病毒感染的早期诊断和疫情的早期控制具有重要意义,适合于基层医疗机构常规应用。     

乙肝病毒前S1抗原时间分辨荧光免疫分析试剂盒的研制

目的利用时间分辨荧光免疫分析（TRFIA）技术建立乙型肝炎病毒前S1抗原的检测试剂盒。方法应用双抗体夹心法建立乙肝preS1抗原TRFIA检测试剂盒,对试剂盒的各项性能指标进行评估。结果对280份血清样本进行检测并与国产酶联免疫法试剂盒对比,结果显示自制试剂盒的特异性更好：200份正常人血清样本检测结果表明,该试剂盒的cutoff值=阴性对照荧光值×4．5。用自制质控品检测分析内和分析间的精密度分别为3．4％～7．8％,6．9％-8.4％;检测HBsAg及HBeAg无交叉反应。结论试剂盒各项指标均达到临床检测要求,TRFIA法精密度和特异性有较大提高,可替代酶免法试剂盒。     

Clinical Evaluation of Rapid Diagnostic Test Kit Using the Polysaccharide as a Genus-Specific Diagnostic Antigen for Leptospirosis in Korea,Bulgaria, and Argentina

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.     

Baculovirus Expression of the Fusion Protein Gene of Bovine Respiratory Syncytial Virus and Utility of the Recombinant Protein in a Diagnostic Enzyme Immunoassay

The fusion (F) protein of bovine respiratory syncytial virus (BRSV) was expressed by using a baculovirus vector. Antigenicity was tested by immunofluorescence analysis with F-specific monoclonal and polyclonal antibodies. Antibodies to recombinant F protein raised in a rabbit neutralized BRSV and human respiratory syncytial virus infectivity when tested in a plaque reduction assay. The recombinant F protein was evaluated as a source of antigen in an enzyme-linked immunosorbent assay (ELISA), and this ELISA was compared with the virus neutralization (VN) test for detecting BRSV antibodies in 10 consecutive serum samples from four calves vaccinated with a live modified BRSV vaccine and from two nonvaccinated control calves. The ELISA with the baculovirus-expressed F protein as an antigen compared favorably with the VN test and is a rapid, sensitive, and specific method for detecting serum antibodies to BRSV.     

Comparison of PanBio Dengue Duo Enzyme-Linked Immunosorbent Assay (ELISA) and MRL Dengue Fever Virus Immunoglobulin M Capture ELISA for Diagnosis of Dengue Virus Infections in Southeast Asia

The performances of the MRL dengue fever virus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the PanBio Dengue Duo IgM capture and IgG capture ELISA were compared. Eighty sera from patients with dengue virus infections, 24 sera from patients with Japanese encephalitis (JE), and 78 sera from patients with nonflavivirus infections, such as malaria, typhoid, leptospirosis, and scrub typhus, were used. The MRL test showed superior sensitivity for dengue virus infections (94 versus 89%), while the PanBio test showed superior specificity for JE (79 versus 25%) and other infections (100 versus 91%). The PanBio ELISA showed better overall performance, as assessed by the sum of sensitivity and specificity (F value). When dengue virus and nonflavivirus infections were compared, F values of 189 and 185 were obtained for the PanBio and MRL tests, respectively, while when dengue virus infections and JE were compared, F values of 168 and 119 were obtained. The results obtained with individual sera in the PanBio and MRL IgM ELISAs showed good correlation, but this analysis revealed that the cutoff value of the MRL test was set well below that of the PanBio test. Comparing the sensitivity and specificity of the tests at different cutoff values (receiver-operator analysis) revealed that the MRL and PanBio IgM ELISAs performed similarly in distinguishing dengue virus from nonflavivirus infections, although the PanBio IgM ELISA showed significantly better distinction between dengue virus infections and JE. The implications of these findings for the laboratory diagnosis of dengue are discussed.     

New Sensitive Competitive Enzyme-Linked Immunosorbent Assay Using a Monoclonal Antibody against Nonstructural Protein 1 of West Nile Virus NY99

An anti-West Nile virus (anti-WNV) monoclonal antibody, SHW-7A11, was developed for competitive enzyme-linked immunosorbent assays (c-ELISAs). SHW-7A11 reacted with nonstructural protein 1 in Western blot analysis. SHW-7A11 was relatively specific for the WNV strain NY99 and recognized Kunjin and Eg101 strains in indirect ELISAs. Two c-ELISAs were developed for sera diluted 10 and 100 times and named c-ELISA10 and c-ELISA100, respectively. Both c-ELISAs detected antibodies against WNV NY99 and Kunjin strains. Little cross-reactivity was observed for antibodies against Japanese encephalitis virus and St. Louis encephalitis virus in these assays. Using the cutoff point for the St. Louis encephalitis virus, all WNV-infected chickens were found to be positive on day 21 after infection in both c-ELISAs. On the other hand, all infected chickens were found to be positive on day 35 after infection in a virus neutralization test. Our newly developed SHW-7A11-based c-ELISA can detect WNV infection with sera diluted 10 to 100 times. Therefore, this c-ELISA can be used for WNV serosurveillance of chickens and wild birds.     

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.     

Evaluation of a New Commercially Available Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Dengue Virus Infection

We evaluated the FDA-cleared InBios dengue virus (DENV) IgM capture enzyme-linked immunosorbent assay (ELISA) for qualitative detection of anti-DENV IgM antibodies from 79 serum samples obtained from dengue virus-infected patients or suspected dengue cases. The agreement, sensitivity, and specificity of the InBios assay compared to the gold standard in-house DENV IgM capture ELISA were 94, 92, and 94%, respectively. We conclude that the InBios DENV IgM capture ELISA can be effectively used for rapid diagnosis of acute or recent DENV infection.     

Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

Antigen capture enzyme-linked immunosorbent assay (ELISA) is one of the most useful methods to detect Ebola virus rapidly. We previously developed an antigen capture ELISA using a monoclonal antibody (MAb), 3-3D, which reacted not only to the nucleoprotein (NP) of Zaire Ebola virus (EBO-Z) but also to the NPs of Sudan (EBO-S) and Reston Ebola (EBO-R) viruses. In this study, we developed antigen capture ELISAs using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques.     

Diagnosis of Dengue Virus Infection by the Visual and Simple AuBioDOT Immunoglobulin M Capture System

The Dengue IgM Capture ELISA (MAC-ELISA) is the immunoenzymatic system recommended by the Pan American Health Organization and the World Health Organization for the serological diagnosis of dengue virus infection due to its high sensitivity, ease of performance, and use of a single acute-phase serum sample. However, tests with this enzyme-linked immunosorbent assay (ELISA) system are time-consuming and require equipment for washing, incubation, and reading of the results. AuBioDOT is a multistep visual diagnostic immunoassay that uses technology based on the immunoglobulin M (IgM) capture ELISA principle. This system uses white polyethylene opaque plates as the solid phase, colloidal gold as the marker, and silver ion amplification. It does not require special equipment, it is totally manually operated, and it can be performed in less than 1 h. The sensitivity and specificity of AuBioDOT for the detection of anti-dengue virus IgM antibodies were studied with a panel of 336 serum samples (150 serum samples from patients with suspected or serologically confirmed dengue virus infection, 186 serum samples from healthy blood donors and patients without dengue virus infection). The results were compared with those obtained by the MAC-ELISA. A sensitivity of 97.7% and a specificity of 97.1% were obtained. The concordance of the two tests was 97.3%, with a kappa index of 0.94. The application of AuBioDOT for the detection of anti-dengue virus IgM antibodies is recommended as an alternative method for the diagnosis of dengue virus infection, both for clinical diagnosis and for seroepidemiological surveillance. The system is useful under field conditions and in laboratories and requires little equipment.     

Genetic Vaccination of Mice with Plasmids Encoding the NS1 Non-structural Protein from Tick-borne Encephalitis Virus and Dengue 2 Virus

Although there is a safe, inexpensive and efficacious vaccine against yellow fever, vaccination against other flavivirus diseases is less successful. There is no licensed vaccine against dengue fever and current vaccines against tick-borne encephalitis (TBE) and Japanese encephalitis are expensive and require several injections. Furthermore novel vaccines containing only virus envelope proteins may raise fears over antibody mediated enhancement (ADE) of disease. Here we report the successful use of genetic vaccination against TBE in an experimental animal model using a plasmid containing the coding sequence of a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to heterotypic infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of the virus NS1 protein normally, no protection was observed against either TBE or dengue fever.