| The mosquito-borne dengue disease is caused by infections of the dengue virus (DENV). It is a major public health problem that affects over 100 tropical and subtropical countries. Moreover, the geographical territory affected by this disease has increased markedly since the 1970s; contributing factors may include a lack of control over the mosquito vector, global warming, viral evolution, and increased ease of global travel. The dengue virus has four known serotypes (DENV1, DENV2. DENV3, and DENV4). An infection with any of these serotypes can cause dengue fever, dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS). Subsequent heterologous infections may increase the risk of the developing DHF or DSS. Since protection against heterologous serotype infections is only partial and transient, people that live in epidemic areas are at risk for contracting an average of four DENV infections in their lives. Thus, the cocirculation of various DENV serotypes in a community is the most common risk factor associated with the emergence of the severe forms of disease (DHF or DSS). Given the current absence of a vaccine that is effective against all four DENV serotypes, early diagnosis is the key to reducing the morbidity and mortality of DHF or DSS. Furthermore, symptoms of DENV infections are insufficiently specific for accurate clinical differentiation from other acute febrile illnesses, especially in areas where multiple tropical diseases such as malaria, yellow fever, West Nile disease, and St Louis encephalitis are endemic. Case identification has become increasingly important in the differential diagnosis of the acute febrile illnesses and hemorrhagic fever of unknown flavivirus infections during the early symptomatic phase. Currently, molecular techniques (reverse transcription-PCR[RT-PCR]) have widely replaced traditional virus isolation methods in the early detection of DENV infections and differentiation of the four DENV serotypes and other related flavivirus infections. Although molecular detection provides a promising sensitivity rate and rapid diagnosis, the molecular approach is costly, as it requires specialized laboratory equipment and experienced technicians; these are notable limitations in many developing countries where dengue is endemic.An 'ideal' simple, cost-effective, rapid, and accurate diagnostic tool is necessary to overcome the main disadvantage of current molecular techniques. Viral antigen detection methods that are convenient and cost-effective have been successfully used in various infectious diseases for the diagnosis and monitoring of disease activity. DENV contains three structural (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Among these proteins, NS1 is a relatively conserved 45- to 50-kDa glycoprotein that is highly expressed in infected cells in both membrane-associated and -secreted forms. Although the precise function of the NS1 is unknown, it represents an interesting target antigen for diagnosis . Recently, two DENV NS1 antigen capture immunoassays have become commercially available for the early diagnosis of dengue virus. The two NS1 antigen capture assays are suitable sensitive and provide a rapid and reliable diagnosis of acute primary dengue disease and acute secondary dengue disease. However, one limitation of these assays is that they are unable to distinguish between the DENV serotypes, as the antibodies used in the assay is directed against cross-reactive antigenic determinants shared by all four DENV serotypes and other flaviviruses. NS1 has been identified as being a both group-specific and type-specific determinant; thus, the production of epitope-specific monoclonal antibodies (MAbs) holds potential for developing either group-specific or type-specific NS1 antigen assays. In a previously study, we prepared a panel of MAbs that recognized epitopes specific to DENV1 NS1 and successfully developed a DENV1 NS1 antigen capture ELISA. This DENV1-specific NS1 antigen assay has proved to be a valuable tool for the early detection and rapid identification of DENV1 infections. In the present study, we prepared a panel of MAbs that recognized epitopes specific to DENV2 NS1 and described the development of a DENV2-specific NS1 antigen capture ELISA.Three parts are included in this research.Part one: Preparation and characterization of monoclonal antibodies against DENV2 NS1 proteinBALB/c mice were immunized by recombinant DENV2 NS1(rDENV2-NS1) and inactivated DENV2. Then the splenocytes of the immunized mice were fused with myeloma cells to produce hybridoma cell line, which could secrete anti-DENV2 NS1 protein antibodies. A total of 37 hybridoma cell lines that stably produced MAbs were initially established on the basis of their strong positive reactivity with both rDENV2-NS1 protein and DENV2-infected cell lysates in the ELISA. The immunoglobulin isotype determinations revealed that the 37 MAbs comprised IgG2a (n=1). IgG2b (n=3). and IgG1 (n=33). Western blot analysis with rDENV2-NS1 protein and DENV2-infected cell culture supernatants revealed that all MAbs reacted to the same immunoreactive protein band with an approximate molecular weight of 45 kDa. The serotype specificity of the MAbs was further identified by testing their reactivity to the four dengue serotypes in the IFA and the rDENV2-NS1-based ELISA. Of the 37 MAbs. 20 MAbs specifically reacted to DENV2 with no cross reactivity to the other three dengue serotypes in either the ELISA or the IFA assays. The remaining 17 MAbs had cross reactivity to the four dengue serotypes in different cross-reactivity patterns. Next, the distinct binding epitopes of the 20 DENV2 NS1-specific MAbs were determined by competition ELISA using rDENV2-NS1 as the immobilized antigen. The results showed that the 20 MAbs bound to at least four different epitopes on the DENV2 NS1 protein. These obtained antibodies will provide a potential value for early diagnosis of DENV infection.Part two: Establishment of the antigen capture ELISA for detection DENV2 NS1 protein. Among the 20 MAbs, each was paired according to their abilities to recognize distinct epitopes on NS1.Then, a sandwich ELISA was performed to select the optimal capture-detector MAb pair. Pairing between eight MAbs in epitope group III and nine MAbs in epitope group IV displayed high sensitivity in DENV2 NS1 protein detection. Ultimately, the pairing of MAb M6 as a solid-phase immobilized capture antibody and MAb M14 as a labeled detecting antibody gave the highest combination of sensitivity and specificity in the detection of DENV2-specific NS1 in serial dilutions of DENV2-infected cell culture supernatants and a panel of sera from healthy humans. The minimal amount of rDENV2-NS1 detection with this assay was approximately 3ng/ml. The linear portion of the standard curve ranged from 10 to 100 ng/ml and could be used to estimate the NS1 levels in clinical samples. Furthermore, the DENV2 NS1 ELISA is specific to DENV2 and has no cross-reactivity with either other serotypes of DV or other closely related members of the flavivirus family (Japanese encephalitis and Yellow fever) when the virus-infected cell culture was used. All these results demonstrated that the established test is sensitive and specific which may lay the basis for providing a novel laboratory diagnosis tool for dengue virus infection.Part three: The preliminary application study of the DENV2 NS1 antigen capture ELISA for detection DENV2 NS1 protein in sera specimens.We compared the sensitivities of the DENV2 NS1 ELISA with the Panbio NS1 ELISA in the detection of NS1 in the DENV2- infected culture supernatants with serial dilutions. Based on the cutoff values of the two assays, the highest dilution of detectable NS1 was 1:4096 (5.8 PFU/0.1mL) in the DENV2 NS1 ELISA, while the highest dilution of detectable NS1 was 1:512 (46.8 FU/0.1mL) in the Panbio NS1 ELISA. Thus, the DENV2 NS1 ELISA demonstrated an approximately 8-fold higher sensitivity than the Panbio NS1 ELISA. Serum specimens from 504 healthy humans were analyzed to establish the background signals of the DENV2 NS1 ELISA before further clinical evaluation. We found that no false positive results were obtained with any of the samples from the 504 sera, indicating the specificity of the DENV2 NS1 ELISA was 100% (0 of 504). To evaluate the accuracy of the DENV2 NS1 ELISA for the detection of NS1 from clinical serum samples, we assessed a total of 30 acute-phase serum specimens from DENV2-infected patients with laboratory-confirmed. Among these sera, 10 were confirmed by both DENV2 isolation and RT-PCR results and 20 were confirmed by both RT-PCR results and Panbio Dengue IgM or IgG capture ELISAs. According to World Health Organization criteria, 17 sera were serologically classified as acute primary infections and 13 sera classified as secondary infections. Based on a sample dilution of 1:10. the DENV2 NS1 ELISA detected 25 of the 30 acute-phase serum specimens. The overall sensitivity of DENV2 NS1 antigen detection with respect to virus isolation and/or RT-PCR was 83.3% (25 of 30). These results were consistent with the results from the Panbio NS1 ELISA when the assays were performed at a 1:10 dilution in parallel: the sensitivities for both assays were 83.3% (25 of 30). of which 5 did not have a detectable NS1 in both the assays. Both NS1 ELISA detected 88.2 % (15/17) of the acute primary and 76.9% (10/13) of the acute secondary infections. There was no statistically significant difference in NS1 detection rate between acute primary infections and acute secondary infections (p = 0.628). To further evaluate the sensitivities of both NS1 detection assays, serial dilutions of positive sera were analyzed. From a total of 25 sera detected NS1 antigen positive in both the assays, only 19 samples had sufficient residual volume to allow us to further compare the sensitivity of both NS1 detection assays. Since the DENV2 NS1 assay displayed a marked sensitivity in the DENV2-infected culture supematants as described above, it was not unexpected that DENV2 NS1 remained detectable even when patient's serum was at a 1:10,000 dilution.DENV2 NS1 ELISA was also evaluated on a panel of clinical serum samples from patients infected with DENV1 or other flaviviruses and non-flaviviruses. We analyzed a total of 301 acute-phase serum samples that had previously tested positive for NS1 by the DENV1 NS1 antigen capture ELISA. Additionally, we tested a total of 132 acute-phase serum samples, comprising 50 samples from 50 cases of hemorrhagic fever with Hantan virus infections. 13 samples from 13 cases of Japanese encephalitis. 49 samples from 49 cases of measles, and 20 samples from 18 cases of leptospirosis. We found that none of these samples were positive for NS1 by the DENV2 NS1 ELISA. These findings suggested that the serotype-specific MAb-based NS1 antigen-capture ELISA might be a valuable tool for early diagnosis and serotyping of DV infections.In conclusion,1. A panel of MAbs specific to the DENV2 NS1 was successfully obtained. This may provide a potential source for studying the function of NS1 and early diagnosis of dengue virus infections.2. A MAb-based antigen capture ELISA for detection circulating NS1 protein was successfully established. This assay was displayed higher sensitivity than commercially available dengue NS1 antigen-capture ELISA. The high detection sensitivity makes it suitable for the early detection of DENV infections.3. A DENV2-specific MAb-based antigen-capture ELISA has been developed. It has high detection specificity to DENV2 without cross-reactions with either other three DENV serotypes or other closely related members of the flavivirus. The characteristic will be helpful in rapid serotyping and differential diagnosis of dengue virus infections.
|
PDF Full Text Request