Kun Wen

Sensitive and Specific Monoclonal Antibody-Based Capture Enzyme Immunoassay for Detection of Nucleocapsid Antigen in Sera from Patients with Severe Acute Respiratory Syndrome

ABSTRACT 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.

Kinetics of non-structural protein 1, IgM and IgG antibodies in dengue type 1 primary infection

BackgroundEarly and accurate diagnosis of dengue infection is essential for control of disease outbreaks. Recently, the dengue virus non-structural antigen 1 (NS1), a conserved and secreted glycoprotein, has been used as a marker for early diagnosis of dengue with convenience and cost-effectiveness. Serological tests of dengue IgM and IgG antibodies are still the most widely used for diagnosis of dengue. In order to assess combined diagnostic value of these tests, we study the kinetic profiles of circulating NS1, dengue IgM and IgG antibodies over the course of the disease by using an in-house dengue type 1 (DENV1) specific NS1 capture ELISA and the commercial Panbio Dengue IgM and IgG capture ELISAs.ResultsA panel of 313 acute-and early convalescent-phase serum specimens from 140 DENV1 primary infected patients during an outbreak of dengue in Guangzhou, China, in 2006 were studied. Dengue NS1 presented high levels in acute-phase serum samples. It was detectable as early as day 1 of illness, and up to 14 day after onset. The sensitivity of NS1 detection was ranged from 81.8% to 91.1% with samples taken during the first 7 days. Anti-dengue IgM antibody was detectable on the third day of onset with the positive rate of 42.9%, and rapidly increasing to 100% by day 8 of illness. Anti-dengue IgG antibody was detectable on the fifth day of onset with low level at the first week of onset, and slowly increasing to 100% by day 15 of illness. Combining the results of NS1 and IgM antibody detection allowed positive diagnosis in 96.9% -100% for samples taken after day 3 of onset.ConclusionsDengue NS1 detection might shorten the window period by first few days of illness. A combination of dengue NS1 antigen and IgM antibody testing facilitates enhanced diagnosis rates. The procedures should be suitable for developing countries where dengue is endemic.

Full Serotype- and Group-Specific NS1 Capture Enzyme-Linked Immunosorbent Assay for Rapid Differential Diagnosis of Dengue Virus Infection

ABSTRACT Dengue virus (DENV), a member of the Flavivirus family, has four distinct serotypes (DENV serotype 1 [DENV1], DENV2, DENV3, and DENV4) that require differentiation for the effective prevention of morbid disease. Early and rapid differentiation between flaviviruses remains challenging. Full assays combining four individual, serotype-specific and one group-specific nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MAbs) against DENV NS1 were developed and validated. The sensitivities and specificities of the full NS1 ELISAs were evaluated with viral cultures and dengue acute-phase sera. Four serotype-specific NS1 ELISAs displayed high specificities for the detection and differentiation of appropriate serotypes. The group-specific NS1 ELISA was broadly reactive with the four dengue virus serotypes. None of the NS1 ELISAs displayed cross-reactivity with the other flaviviruses or samples from febrile patients with non-dengue virus infections. The full serotype- and group-specific MAb-based NS1 capture ELISAs may provide tools for the early detection and typing of dengue infection, which is preferable to reverse transcriptase PCR (RT-PCR) for the rapid differential diagnosis of dengue virus infection in the field.

Antigenic Cross-Reactivity between Severe Acute Respiratory Syndrome—Associated Coronavirus and Human Coronaviruses 229E and OC43

Abstract 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 ⩾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.

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

ABSTRACT 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.

Comparison of plaque- and enzyme-linked immunospot-based assays to measure the neutralizing activities of monoclonal antibodies specific to domain III of dengue virus envelope protein.

ABSTRACT The plaque reduction neutralization test (PRNT) is used widely to measure the neutralization activity of anti-dengue virus (DENV) antibodies, but it is time-consuming and labor-intensive and has low sample throughput. For fast and convenient measurement of neutralizing antibodies, especially in evaluating the efficiency of the DENV vaccines on a large scale, a new method is needed to replace PRNT. In recent decades, several microneutralization assays have been developed to overcome the limitations of PRNT. In the present study, we evaluated one of these, the enzyme-linked immunospot microneutralization test (ELISPOT-MNT), in comparison with PRNT. ELISPOT-MNT is performed in 96-well format, and the plaques are developed after 2 to 4 days using an ELISA to transform them into spots, which are detected automatically with an ELISPOT instrument. The assay is faster than PRNT, has a high throughput, and is more objective. We used 10 monoclonal antibodies (MAbs) against domain III of the DENV envelope protein (EDIII) to evaluate the two assays; all of these MAbs cross-react with all four serotypes of DENV as measured by immunofluorescence assay. The two neutralization assays were performed simultaneously to measure the 50% inhibitory concentration (IC50) of these MAbs. Using PRNT as the reference and treating IC50 values higher than 50 μg/ml of MAbs as negative, ELISPOT-MNT showed a sensitivity of 95.6% and specificity of 88.24% when 10 MAbs were tested against four DENV serotype strains. A good correlation (R2 = 0.672; P = 0.000) was observed between the two assays, making ELISPOT-MNT a potentially valuable method for measure of neutralizing antibodies against DENV.

CROSS-REACTIVITY OF ANTI-EV71 IgM AND NEUTRALIZING ANTIBODY IN SERIES SERA OF PATIENTS INFECTED WITH ENTEROVIRUS 71 AND COXSACKIEVIRUS A 16

Objectives: To evaluate the cross-reactivity of anti-EV71 IgM and neutralizing antibody in series sera of patients infected with EV71 and CA16. Methods: Real-time RT-PCR, virus isolation, ELISA and neutralization test were used to detect enteroviruses from clinical specimens and series sera of 79 HFMD patients. Results: 27 EV71, 37 CA16, and 11 other enterovirus-infected patients were identified by RT-PCR. Among EV71 infected patients, anti-EV71 IgM positive ratios were 87.5% during 1–3 days after onset and 100% over 4 days after onset. In CA16 infected patients, the positive ratios were 7.4%, 26.4%, and 62.5% during 1–3 days, 4–6 days, and over 6 days after onset, respectively. Meanwhile, the results of neutralization test showed 18.9% of CA16 infected patients and 11.1% of EV71 infected patients present high cross-neutralization antibody against each other. Conclusions: Cross-reactivity of anti-EV71 IgM in patients infected with EV71 and CA16 becomes stronger with the progress of disease. Moreover, high cross-neutralization antibody existing in part of patients suggests that the immune reactivity to EV71infection can be recalled by CA16, and the immune reactivity to CA16 infection can be recalled by EV71. Therefore, identifying enteroviruses by neutralization test may not be an ideal selection.

Monoclonal Antibody-Based Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Sensitivity of the Nucleocapsid Protein in Acute-Phase Sera of Severe Acute Respiratory Syndrome Patients

ABSTRACT Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum samples from healthy donors and non-SARS febrile patients. The results of the N protein detection analysis were directly related to the serological analysis data. From 27 SARS patients who tested positive with the neutralization test, 100% of the 24 sera collected from 1 to 10 days after the onset of symptoms were positive for the N protein. N protein was not detected beyond day 11 in this group. The positive rates of N protein for sera collected at 1 to 5, 6 to 10, 11 to 15, and 16 to 20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients were 92.9, 69.8, 36.4, and 21.1%, respectively. For 294 sera from 248 serological test-negative patients, the rates were 25.6, 16.7, 9.3, and 0%, respectively. The N protein was not detected in 66 patients with cases of what was initially suspected to be SARS but serologically proven to be negative for SARS and in 197 serum samples from healthy donors and non-SARS febrile patients. The specificity of the assay was 100%. Furthermore, of 16 sera collected from four patients during the SARS recurrence in Guangzhou, 5 sera collected from 7 to 9 days after the onset of symptoms were positive for the N protein. N protein detection exhibited a high positive rate, 96 to 100%, between day 3 and day 5 after the onset of symptoms for 27 neutralization test-positive SARS patients and 298 serologically confirmed patients. The N protein detection rate continually decreased beginning with day 10, and N protein was not detected beyond day 19 after the onset of symptoms. In conclusion, an antigen capture ELISA reveals a high N protein detection rate in acute-phase sera of patients with SARS, which makes it useful for early diagnosis of SARS.

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.

Functional properties of DENV EDIII‑reactive antibodies in human DENV‑1‑infected 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 anti‑EDIII antibodies contribute little to the neutralizing or enhancing ability of human DENV‑infected serum. The present study involved an analysis of the neutralization and antibody‑dependent enhancement (ADE) activities of EDIII‑reactive antibodies in human convalescent sera from patients with primary DENV‑1 infection and rabbit antiserum immunized with recombinant DENV‑1 EDIII protein. The results indicated that serum neutralization was not associated with titres of EDIII‑binding antibodies in the human DENV‑1‑infected sera. The depletion of anti‑EDIII antibodies from these serum samples revealed that the anti‑EDIII antibodies of the patients contributed little to neutralization and ADE. However, the EDIII‑reactive 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 EDIII‑reactive 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.