Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling

Abstract

Dengue fever, a mosquito-borne disease in tropical and subtropical climates caused by the dengue virus (DENV), has become a major social and economic burden in recent years. However, current primary detection methods are inadequate for early diagnosis of DENV because they are either time-consuming, expensive, or require training. Non-structural protein 1 (NS1) is secreted during DENV infection and is thus considered a suitable biomarker for the development of an early detection method. In the present study, we developed a detection method for the NS1 protein based on a previously reported thio-NAD cycling ELISA (i.e., ultrasensitive ELISA) and successfully achieved a LOD of 1.152 pg/mL. The clinical diagnosis potential of the detection system was also evaluated by using 85 patient specimens, inclusive of 60 DENV-positive and 25 DENV-negative specimens confirmed by the NAAT method. The results revealed 98.3% (59/60) sensitivity and 100% (25/25) specificity, which was in almost perfect agreement with the NAAT data with a kappa coefficient of 0.972. The present study demonstrates the diagnostic potential of using an ultrasensitive ELISA as a low-cost, easy-to-use method for the detection of DENV compared with NAAT and could be of great benefit in low-income countries.

Keywords: NS1 protein; dengue virus; detection; nucleic acid application test; ultrasensitive ELISA.

Figure 1

Illustration of ultrasensitive ELISA with thio-NAD cycling. A pair of antibodies was used for capturing the NS1 protein in the sandwich ELISA, and alkaline phosphatase was labeled on the secondary antibody. Aside from the antibodies, an androsterone derivative, 3α-hydroxysteroid dehydrogenase, thio-NAD, and NADH were used to construct the thio-NAD enzyme cycling system. During the thio-NAD cycling reaction, thio-NADH constantly accumulated in a triangular number fashion and could be directly measured at an absorbance of 405 nm.

Figure 2

Linear calibration curve obtained from the absorbance for the recombinant DENV type 2 NS1 protein using the thio-NAD cycling ELISA. The absorbance was obtained from the cycling reaction time designated in the text. The experiment was performed with four sets of wells. The antigen was applied in the range of 12.5–200 pg/mL. See Table S2: Raw data of Figure 2 in Supplementary Materials for more detailed information.