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.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression and purification of recombinant DV1 NS1 protein from Escherichia coli. Lane 1, uninduced crude cell lysates; lanes 2 to 5, induced crude cell lysates under different conditions; lanes 6 to 9, recombinant DV1 NS1 protein purified by Ni-nitrilotriacetic acid affinity chromatography; M, markers with their corresponding molecular masses.

FIG. 2.

Sensitivity of sandwich formations in antigen capture ELISA from different pairs of capturer and detector MAbs determined by replicates of several dilutions of DV1 culture filtrates.

FIG. 3.

Standard curve for NS1 determined with purified recombinant DV1 NS1 protein by antigen capture ELISA. Various concentrations of DV1 NS1 protein were analyzed. BSA was used to establish the baseline. Data points represent means ± standard deviations for 10 replicates.

FIG. 4.

Evaluation of the sensitivity and specificity of the DV1 NS1 antigen capture ELISA for detection of the DV1 NS1 protein in flaviviruses and nonflavivirus cultures. Serial dilutions of virus culture filtrates that were obtained from the four serotypes of DV, JEV, and YFV were subjected to the DV1 NS1 antigen capture ELISA. The positive OD₄₅₀ values are restricted to NS1 of serotype DV1.

FIG. 5.

Evaluation of the sensitivity and specificity of the DV1 NS1 antigen capture ELISA for detection of the DV1 NS1 protein in different serum samples. Data represent the OD₄₅₀ of sera tested at a dilution of 1/3. Serum samples were taken from the following sources: A, clinically probable DV1-infected patients during the DV1 epidemic in Guangdong in 2002 and 2003 (n = 462); B, DV1-infected patients identified by serotyping RT-PCR (n = 17); C, 20 documented DV2-infected patients and one DV3-infected patient; D, JEV-infected patients (n = 13); E, Hantan virus-infected patients (n = 51); F, measles virus-infected patients (n = 56); G, patients with leptospirosis (n = 20); H, healthy blood donors (n = 469).

FIG. 6.

Profile of DV1 NS1 detection in blood circulation and IgM antibody response to DV from the onset of symptoms to the convalescence phase.