Changes in enterovirus serotype constituent ratios altered the clinical features of infected children in Guangdong Province, China, from 2010 to 2013.

BACKGROUND: Enterovirus (EV)-related hand, foot, and mouth disease/herpangina (HFMD/HA) has been prevalent in Guangdong Province, China, since 2010. METHODS: Clinical data for EV-related HFMD/HA inpatients admitted to the Department of Paediatrics of Zhujiang Hospital from 2010 to 2013 were retrospectively reviewed. The corresponding EV serotypes were also determined by reverse transcription-polymerase chain reaction or BLAST analysis of the sequenced partial lengths of the viral protein1/5'-untranslated region. RESULTS: A total of 867 eligible inpatients admitted during 2010-2013 were included in the study. Of these, the serotype of the responsible EV was successfully identified in 824 cases. The incidence of enterovirus 71 (EV71) infection amongst pediatric HFMD/HA inpatients decreased dramatically from 55.5 % in 2010 to 8.1 % in 2013, with a similar decrease recorded for coxsackievirus A16 (CVA16). However, the incidence of non-EV71/CVA16 infection increased from 30.0 % in 2010 to 83.8 % in 2013. We noted that the types of infection caused by different EV serotypes varied: EV71 was responsible for 100 % of the paralysis cases (26/26), 84.6 % of the deaths (11/13), and 84.1 % of cases with severe central nervous system involvement (SCNSI) (74/88); echovirus contributed to 16.4 % of the deaths (2/13) and 4.4 % of the SCNSI cases; and coxsackievirus accounted for only 2.2 % of the SCNSI cases (2/90). The clinical features of HFMD/HA cases varied greatly during the time period examined, with drastic changes in the hospitalization rates (45.1, 63.7, 36.4, and 19.1 % for 2010, 2011, 2012, and 21013, respectively), mortality rates (2.3, 0.9, 2.5, and 0.0 %, respectively), paralysis (5.1, 1.2, 5.4, and 0.0 %, respectively), SCNSI (16.8, 7.1, 12.7, and 2.2 %, respectively), and acute respiratory infection (21.1, 22.0, 45.9, and 59.0 %, respectively). CONCLUSIONS: The incidences of infection caused by different EV serotypes, along with the clinical features of HFMD/HA cases, changed drastically in Guangdong Province, China, from 2010 to 2013, with the biggest changes observed in 2013. The changed constituent ratios of the different EV serotypes might therefore be responsible for the differences in the observed clinical features of HFMD/HA during this period.

Functional properties of DENV EDIII­reactive antibodies in human DENV­1­infected sera and rabbit antiserum to EDIII.

The envelope domain III (EDIII) of the dengue virus (DENV) has been confirmed to be involved in receptor binding. It is the target of specific neutralizing antibodies, and is considered to be a promising subunit dengue vaccine candidate. However, several recent studies have shown that anti­EDIII antibodies contribute little to the neutralizing or enhancing ability of human DENV­infected serum. The present study involved an analysis of the neutralization and antibody­dependent enhancement (ADE) activities of EDIII­reactive antibodies in human convalescent sera from patients with primary DENV­1 infection and rabbit antiserum immunized with recombinant DENV­1 EDIII protein. The results indicated that serum neutralization was not associated with titres of EDIII­binding antibodies in the human DENV­1­infected sera. The depletion of anti­EDIII antibodies from these serum samples revealed that the anti­EDIII antibodies of the patients contributed little to neutralization and ADE. However, the EDIII­reactive antibodies from the rabbit antiserum exhibited protective abilities of neutralization at a high dilution (~1:50,000) and ADE at a low dilution (~1:5,000) for the homotypic DENV infection. Notably, the rabbit antiserum displayed ADE activity only at a dilution of 1:40 for the heterotypic virus infection, which suggests that EDIII­reactive antibodies may be safe in secondary infection with heterotypic viruses. These results suggest that DENV EDIII is not the predominant antigen of the DENV infection process; however, purified or recombinant DENV EDIII may be used as a subunit vaccine to provoke an effective and safe antibody response.

Enterovirus-related diarrhoea in Guangdong, China: clinical features and implications in hand, foot and mouth disease and herpangina.

BACKGROUND: A series of complications caused by enteroviruses, including meningitis, encephalitis, acute flaccid paralysis, acute cardiopulmonary failure, respiratory infection, and myocardial injury have been reported in hand, foot and mouth disease/herpangina (HFMD/HA). However, the complication of diarrhoea caused by enteroviruses has been neglected, and a summary of its clinical features and impact on HFMD/HA is unavailable. METHODS: We included inpatients with HFMD/HA admitted to the Paediatric Department of Zhujiang Hospital during 2009-2012. We summarised and compared clinical data for cases with and without diarrhoea, and determined enterovirus serotypes by reverse transcriptase polymerase chain reaction and genotyping based on a partial-length fragment of viral protein 1 or the 5'-untranslated region. RESULTS: There were 804 inpatients with HFMD/HA and 28 (3.5%) presented with diarrhoea. Gastrointestinal symptoms were mild in most cases of diarrhoea (82.1%), with high prevalence of no dehydration (82.1%), short duration of diarrhoea (78.6%) and watery stools (75.0%). The prevalence of multi-organ dysfunction syndrome (10.7 vs 0.40%) (p = 0.001), hepatic injury (14.3 vs 3.4%) (p = 0.019), myocardial injury (21.4 vs 6.1%) (p = 0.002) and convulsion (21.4 vs 7.2%) (p = 0.016) was significantly higher in the diarrhoea than no diarrhoea group. There was no significant difference between the two groups regarding prevalence of death, altered consciousness, paralysis, central nervous system involvement, or acute respiratory infection. CONCLUSIONS: Most patients with diarrhoea caused by enteroviruses circulating in Guangdong Province in 2009-2012 had mild or moderate gastrointestinal symptoms. Although enterovirus-related diarrhoea caused additional multi-organ dysfunction syndrome, hepatic injury and myocardial injury in children with HFMD/HA, timely intervention efficiently reduced disease severity and improved outcome.

The absence of exanthema is related with death and illness severity in acute enterovirus infection.

OBJECTIVE: To clarify whether exanthema is related to illness severity in acute enterovirus infection in children. METHODS: The data of pediatric inpatients at Zhujiang Hospital during 2009-2012 with an acute enterovirus infection were reviewed retrospectively. Enterovirus infection was determined by real-time reverse transcription PCR. Clinical data were summarized and compared between cases with and without exanthema. RESULTS: A total of 780 pediatric inpatients with an acute enterovirus infection were included in this study, of whom 83 (10.6%) presented no exanthema. The percentage of deaths in the group of patients without exanthema was significantly higher than that in the group with exanthema (7.2% vs. 1.1%; p = 0.002). Central nervous system involvement (41.0% vs. 30.0%; p = 0.041), severe central nervous system (CNS) involvement (21.7% vs. 11.0%; p = 0.005), severe CNS involvement with cardiopulmonary failure (9.6% vs. 2.3%; p = 0.002), an altered level of consciousness (15.7% vs. 7.6%; p = 0.013), and convulsions (14.4% vs. 6.3%; p = 0.007) occurred significantly more frequently in the group without exanthema. CONCLUSIONS: A considerable proportion of children with an acute enterovirus infection in Guangdong Province, China during 2009-2012 presented no exanthema, and the absence of exanthema was found to be related to death and illness severity for these acute enterovirus infections. Clinicians in China should consider enterovirus as the possible pathogen when treating children with an acute pathogen infection without exanthema.

Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice.

Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3'-untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence.

Dengue virus envelope domain III immunization elicits predominantly cross-reactive, poorly neutralizing antibodies localized to the AB loop: implications for dengue vaccine design.

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime-boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309-320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime-boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.

Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays.

The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose-response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

Evaluation of human enterovirus 71 and coxsackievirus A16 specific immunoglobulin M antibodies for diagnosis of hand-foot-and-mouth disease.

BACKGROUND: Hand-foot-and-mouth disease (HFMD) is caused mainly by the human enterovirus type 71 (HEV71) and the Coxsackievirus A group type 16 (CVA16). Large outbreaks of disease have occurred frequently in the Asia-Pacific region. Reliable methods are needed for diagnosis of HFMD in children. IgM-capture ELISA, with its notable advantages of convenience and low cost, provides a potentially frontline assay. We aimed to evaluate the newly developed IgM-capture ELISAs for HEV71 and CVA16 in the diagnosis of HFMD, and to measure the kinetics of IgM over the course of HEV71 or CVA16 infections. RESULTS: We mapped, for the first time, the kinetics of IgM in HEV71 and CVA16 infection. HEV71- and CVA16-IgM were both detectable in some patients on day 1 of illness, and in 100% of patients by day 5 (HEV71) and day 8 (CVA16) respectively; both IgMs persisted for several weeks. The IgM detection rates were 90.2% (138 of 153 sera) and 68.0% (66 of 97 sera) for HEV71 and CVA16 infections, respectively, during the first 7 days of diseases. During the first 90 days after onset these values were 93.6% (233 of 249 sera) and 72.8% (91 of 125 sera) for HEV71 and CVA16 infections, respectively. Some cross-reactivity was observed between HEV71- and CVA16-IgM ELISAs. HEV71-IgM was positive in 38 of 122 (31.1%) CVA16 infections, 14 of 49 (28.6%) other enteroviral infections and 2 of 105 (1.9%) for other respiratory virus infected sera. Similarly, CVA16-IgM was apparently positive in 58 of 211 (27.5%) HEV71 infections, 16 of 48 (33.3%) other enterovirus infections and 3 of 105 (2.9%) other respiratory virus infected sera. Nevertheless, the ELISA yielded the higher OD450 value of main antibody than that of cross-reaction antibody, successfully identifying the enteroviral infection in 96.6% (HEV71) and 91.7% (CVA16) cases. When blood and rectal swabs were collected on the same day, the data showed that the agreement between IgM-capture ELISA and real-time RT-PCR in HEV71 was high (Kappa value = 0.729) while CVA16 somewhat lower (Kappa value = 0.300). CONCLUSIONS: HEV71- and CVA16-IgM ELISAs can be deployed successfully as a convenient and cost-effective diagnostic tool for HFMD in clinical laboratories.

Enzyme-linked immunosorbent assay-format tissue culture infectious dose-50 test for titrating dengue virus.

A dengue nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA)-based tissue culture infectious dose-50 (TCID(50)) test (TCID(50)-ELISA) was developed as an alternative to the standard plaque assay for titrating dengue virus. Virus titers obtained by TCID(50)-ELISA were comparable to those obtained by the plaque assay and by the traditional TCID(50)-cytopathic effect (CPE) test (TCID(50)-CPE), with a better reproducibility and a lower coefficient of variation. Quantitative comparison of TCID(50)-ELISA and TCID(50)-CPE resulted in a correlation coefficient of 0.976. Moreover, this new method showed a wider application to C6/36, Vero E6, BHK-21, and Vero cells compared with other titration methods. In summary, the novel TCID(50)-ELISA method described here provides a more reliable and more accurate alternative compared to the plaque assay and TCID(50)-CPE for titration of dengue virus.

[To evaluate the neutralizing abilities of anti-dengue virus antibodies with nonstructural protein 1 antigen capture enzyme-linked immunosorbent assay].

OBJECTIVE: To produce neutralizing antibodies against envelope protein domain III (EDIII) of dengue virus serotype I (DENV-1) and evaluate the nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA) for identification of antibody neutralizing abilities. METHODS: Five BALB/c mice and one New Zealand Rabbit were immunized with recombinant EDIII protein of DENV-1 for the production of hybridomas and hyperimmune sera. Indirect ELISA, immunofluorescence assay (IFA) and Western Blot analyses were applied to identify specificity of antibodies. Comparing to plaque reduction neutralization test (PRNT), the new established DENV-1 specific NS1 antigen capture ELISA was used for detecting the neutralizing abilities of these antibody. RESULTS: Four strains of monoclonal antibodies (mAbs) named 1A1, 1B3, 3D3 and 9D6 and one hyperimmune serum of rabbit were obtained, all of which were approved to have neutralizing abilities to DENV-1 with the PRNT titer of 1:1024, 1:512, 1:256, 1:4096 and 1:4096. MAb 3D3 with the lowest neutralization titer in PRNT had not shown neutralizing ability to DENV-1 in NS1 antigen capture ELISA, while MAbs 1A1, 1B3 and 9D6 and the rabbit hyperimmune serum could protect the C6/36 from being infected by DENV-1 with the neutralization titer of 1:32, 1:32, 1:128 and 1:128 in this assay. CONCLUSION: NS1 antigen capture ELISA could be used to identify antibody neutralizing abilities to DENV, it was a faster and more convenient way to screen antibodies with high neutralization titer and might also be used as one of the methods to evaluate the effects of vaccines.

Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections.

The dengue virus (DENV) has four distinct serotypes (DENV1, DENV2, DENV3, and DENV4) that require differentiation for effective prevention of morbid diseases. The recently developed DENV1-specific NS1 antigen capture enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibodies (MAbs) that recognize distinct epitopes on nonstructural protein 1 (NS1) of a specific DENV serotype is convenient and cost-effective, but assays have not yet been developed for DENV serotypes 2 to 4. This paper describes the development and validation of a DENV2-specific NS1 antigen capture ELISA by selection and optimization of the pair of well-characterized MAbs that recognized epitopes specific for DENV2 NS1 from a large panel of MAbs. The DENV2 NS1 ELISA displayed exclusive sensitivity with the DENV2 serotype and did not cross-react with the other three DENV serotypes. The sensitivity and specificity of the DENV2 NS1 ELISA were 83.3% (25/30) and 100% (504/504) when used to test 30 acute-phase serum samples from patients infected with DENV2 identified by virus isolation or reverse transcription-PCR serotyping and 504 serum samples from healthy individuals, respectively. The specificity of this assay was also evaluated using a panel of serum samples which were positive for DENV1, other flaviviruses, and nonflaviviruses; no cross-reactions were observed in these clinical samples. The DENV2 NS1 ELISA was eightfold more sensitive than a commercially available serotype-cross-reactive NS1 ELISA (Panbio Diagnostics, Brisbane, Australia) when the two assays were used to test the DENV2-infected cell culture supernatants in parallel. The Panbio NS1 ELISA displayed variation in sensitivity between DENV serotypes. The DENV2-specific NS1 antigen capture ELISA can be used as a tool for the rapid identification of DENV2 infections.

Well-characterized monoclonal antibodies against cell wall antigen of Aspergillus species improve immunoassay specificity and sensitivity.

The diagnosis of invasive aspergillosis (IA) based on the detection of Aspergillus galactomannan (GM) is complicated by the presence of cross-reactive GM epitopes in patient specimens. We have developed a novel and specific Aspergillus antigen-capture enzyme-linked immunosorbent assay (ELISA) by the selection of two well-characterized monoclonal antibodies from 17 candidate antibodies. The epitopes recognized by the monoclonal antibodies were present on the cell walls of the hyphae and the conidia of Aspergillus species, which were circulating or excreted as immunodominant antigens during the acute phase of IA established in the animal models. The detection of experimental Aspergillus-mediated antigenemia was suitably sensitive, and the sensitivity was comparable to that of a commercial GM detection ELISA kit (the Platelia Aspergillus assay). Moreover, the specificity of this assay was 100% when it was used to test 382 serum specimens and 120 urine specimens from healthy individuals. Cross-reactivity with other common opportunistic fungi, such as Penicillium and Candida species, and with purified GM protein derived from Aspergillus was not evident. Therefore, the chemical nature of the epitopes captured in this assay is most likely not associated with the GM structure, indicating that this newly developed Aspergillus antigen-capture ELISA is a promising tool for the diagnosis of IA without the risk of the false-positive results that are problematic with current GM antigen assays.

Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1: Implications for early diagnosis and serotyping of dengue virus infections.

Rapid diagnosis and serotyping of dengue virus (DV) infections are important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. However, the speed and accuracy of diagnosis must be balanced against test cost and availability, especially in developing countries. We developed a specific antigen capture enzyme-linked immunosorbent assay (ELISA) for early detection and serotyping of DV serotype 1 (DV1) by using well-characterized monoclonal antibodies (MAbs) specific to nonstructural protein 1 (NS1) of DV1. With this assay, a total of 462 serum specimens from clinically probable DV1-infected patients during the DV1 epidemic in Guangdong, China, in 2002 and 2003 were analyzed. DV1 NS1 was detectable in blood circulation from the first day up to day 18 after onset of symptoms, with a peak at days 6 to 10. The sensitivity of DV1 NS1 detection in serum specimens with reference to results from reverse transcriptase PCR was 82%, and the specificity was 98.9% with reference to 469 healthy blood donors. No cross-reactions with any of the other three DV serotypes or other closely related members of the genus Flavivirus (Japanese encephalitis virus and Yellow fever virus) were observed when tested with the clinical specimens or virus cultures. These findings suggest that the serotype-specific MAb-based NS1 antigen capture ELISA may be a valuable tool for early diagnosis and serotyping of DV infections, while also providing a standardized assay for the analysis of a great number of clinical samples with convenience and cost-effectiveness.

Antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229E and OC43.

Cross-reactivity between antibodies to different human coronaviruses (HCoVs) has not been systematically studied. By use of Western blot analysis, indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA), antigenic cross-reactivity between severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and 2 HCoVs (229E and OC43) was demonstrated in immunized animals and human serum. In 5 of 11 and 10 of 11 patients with SARS, paired serum samples showed a > or =4-fold increase in antibody titers against HCoV-229E and HCoV-OC43, respectively, by IFA. Overall, serum samples from convalescent patients who had SARS had a 1-way cross-reactivity with the 2 known HCoVs. Antigens of SARS-CoV and HCoV-OC43 were more cross-reactive than were those of SARS-CoV and HCoV-229E.

Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome.

A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.

[Antibody response of patients with severe acute respiratory syndrome (SARS) to nucleocapsid antigen of SARS-associated coronavirus].

OBJECTIVE: To assess serum antibody responses of patients with severe acute respiratory syndrome (SARS) to nucleocapsid (N) antigen of SARS-associated coronavirus. METHODS: The serum levels of IgM and IgG antibodies to N antigen were measured in 200 healthy blood donors and 13 SARS patients at different time points of acute and convalescent phases using indirect enzyme-linked immunosorbent assay (ELISA) with N fusion proteins of SARS-associated coronaviruses. RESULTS: The IgM positive critical value of 0.233 and IgG of 0.239 were selected as the threshold value for positive results that equals the product of 2.1 and the mean IgM and IgG levels of 200 healthy blood donors. In 13 patients with SARS, the antibody responses to N antigen were not detectable in the first week after the onset of symptoms. The IgM and IgG seroprotection rates were 83.3% and 66.7% respectively in the second week, both reaching 100% at the third week. IgM seroprotection rates was 61.5% in the second month, and 38.5% at third month. The IgG peaked one month after the onset and remained at high levels in the following 2 months. CONCLUSION: The antibody responses suggest that N protein of SARS is immunodominant and plays an important role in viral pathogenesis. This ELISA-based test for detecting anti- N antigen may be of significant value for SARS diagnosis.

Rapid and efficient preparation of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein by immunizing mice.

OBJECTIVE: To develop a rapid and efficient method for preparing monoclonal antibodies (mAb) against SARS-associated coronavirus (SARS-Cov) nucleocapsid (N) protein. METHODS: BALB/c mice were injected with the recombinant N protein of SARS-Cov into the foot-pads for the immunization, and the popliteal lymph nodes were isolated 15 d later for mAb-producing hybridomas, from which the mAbs against the N protein of SARS-Cov were screened. The identification of the mAb against the N protein of SARS-Cov was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. RESULTS: Four strains of hybridomas were obtained that produced the mAb specific to the N protein without detectable cross-reactivity with other pathogens. Of the 4 strains, 2 were identified as the immunoglobulin G1 (IgG1) isotype, 1 IgG2a, and the other IgG2b, with affinity constants (Ka) of 2 of the strains being 4.14 x 10(-9)M and 3.19 x 10(-9)M respectively. CONCLUSION: This is the first report on the preparation of mAb that is specific to the SARS-Cov, and the high-specificity and high-affinity mAb produced by the 4 strains of hybridomas provide a basis for further researches on the pathogenesis and early diagnosis of SARS.