An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients

Abstract

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 microg/ml, were found in acute-phase sera taken from some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.

FIG. 1

Reactivity of a panel of MAbs against serial dilutions of immunoaffinity-purified dengue 2 NS1 in the capture ELISA. NS1 was captured with ELISA plates coated with a 1:1,500 dilution of rabbit anti-NS1 polyclonal antisera and subsequently probed with the MAbs at a dilution of 1:1,000.

FIG. 2

Effect of human serum components on the detection of NS1 in the capture ELISA. Immunoaffinity-purified, secreted NS1 was serially diluted in the absence (●) or presence of NHS at dilutions of 1 in 5 (Δ) and 1 in 10 (○) and probed with the type-specific MAb 1H7.4. Data points represent the mean ± standard deviation for four replicates. The dashed line is the mean for the negative control samples (OD₄₉₀, 0.122 ± 0.012).

FIG. 3

Capture ELISA detection of NS1 secreted from Vero cells infected with all four dengue virus serotypes. Type-specific (1H7.4) (A) and group-reactive (3D1.4) (B) MAbs were used as the detection probes for media harvests from DEN1- (♦), DEN2- (●), DEN3- (■), and DEN4 (▴)-infected cells.