Enzyme-linked immunosorbent assay specific to Dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections

Abstract

During flavivirus infection in vitro, nonstructural protein NS1 is released in a host-restricted fashion from infected mammalian cells but not vector-derived insect cells. In order to analyze the biological relevance of NS1 secretion in vivo, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) to detect the protein in the sera of dengue virus-infected patients. The assay was based on serotype 1 NS1-specific mouse and rabbit polyclonal antibody preparations for antigen immunocapture and detection, respectively. With purified dengue virus type 1 NS1 as a protein standard, the sensitivity of our capture ELISA was less than 1 ng/ml. When a panel of patient sera was analyzed, the NS1 antigen was found circulating from the first day after the onset of fever up to day 9, once the clinical phase of the disease is over. The NS1 protein could be detected even when viral RNA was negative in reverse transcriptase-PCR or in the presence of immunoglobulin M antibodies. NS1 circulation levels varied among individuals during the course of the disease, ranging from several nanograms per milliliter to several micrograms per milliliter, and peaked in one case at 50 microg/ml of serum. Interestingly, NS1 concentrations did not differ significantly in serum specimens obtained from patients experiencing primary or secondary dengue virus infections. These findings indicate that NS1 protein detection may allow early diagnosis of infection. Furthermore, NS1 circulation in the bloodstream of patients during the clinical phase of the disease suggests a contribution of the nonstructural protein to dengue virus pathogenesis.

Figures

FIG. 1.

Standard capture ELISA with purified dengue virus type 1 NS1. NS1 was serially diluted in PBS-Tween buffer. Data points represent the mean and standard deviation for three replicates corrected by the mean value of the negative controls. The cutoff value was set at twice the average value of the negative controls.

FIG. 2.

NS1 antigen capture in the sera of dengue virus type 1-infected patients. Data points represent the OD of sera tested at a dilution of 1/10 and corrected by using the mean value of the corresponding negative controls. Ambiguous sera were tested at 1/2. The numbers of sera with similar ODs are indicated with a number in parentheses near the representatives data point.

FIG. 3.

Daily follow-up of serum-NS1 in dengue virus type 1-infected patients. For each serum, we report the RT-PCR results; the IgM and NS1 ODs, corrected by the mean value of the corresponding negative controls; and the IgM (----) and NS1 (-·-·-·) cutoff values.