Yuxian Pan

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

ABSTRACT 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.

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.

Comprehensive mapping of immunodominant and conserved serotype- and group-specific B-cell epitopes of nonstructural protein 1 from dengue virus type 1

The dengue virus (DENV) nonstructural protein 1 (NS1) is an immunogenic protein that holds potential for the development of vaccines and diagnostic reagents; however, the epitopes of NS1 have not been comprehensively mapped. We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. This strategy identified three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15, 71-85, and 338-352. We also identified five group-specific B-cell epitopes that were highly conserved among isolates of the four DENV serotypes. These novel immunodominant serotype- and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.

Well-Characterized Monoclonal Antibodies against Cell Wall Antigen of Aspergillus Species Improve Immunoassay Specificity and Sensitivity

ABSTRACT 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.

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.

Comparison of surface plasmon resonance, resonant waveguide grating biosensing and enzyme linked immunosorbent assay (ELISA) in the evaluation of a dengue virus immunoassay.

Two label-free biosensor platforms, Resonance Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR), were used to rank a large panel of anti-dengue virus NS1 antibodies. Dengue non-structural 1 (NS1) protein is an established serological marker for the early detection of dengue infection. A variety of commercial dengue NS1 antigen capture immunoassays are available in both ELISA and lateral flow format. However, there is a significant scope to improve both the sensitivity and the specificity of those tests. The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD= 1–0.1 μM) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR. There were significant differences in the absolute affinities determined by the two technologies, and there was a poor correlation between antibodies best ranked by RWG and the lower limit of detection (LLOD) found by ELISA. Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats.