Sensitivity and Cross-Reactivity Analysis of Serotype-Specific Anti-NS1 Serological Assays for Dengue Virus Using Optical Modulation Biosensing

Abstract

Dengue virus (DENV) poses a major global health concern, with over 6.5 million cases and 7300 deaths reported in 2023. Accurate serological assays are essential for tracking infection history, evaluating disease severity, and guiding vaccination strategies. However, existing assays are limited in their specificity, sensitivity, and cross-reactivity. Using optical modulation biosensing (OMB) technology and non-structural protein 1 (NS1) antigens from DENV-1-3, we developed highly sensitive and quantitative serotype-specific anti-DENV NS1 IgG serological assays. The OMB-based assays offered a wide dynamic range (~4-log), low detection limits (~400 ng/L), fast turnaround (1.5 h), and a simplified workflow. Using samples from endemic (Vietnam) and non-endemic (Israel) regions, we assessed intra-DENV and inter-Flavivirus cross-reactivity. Each assay detected DENV infection with a 100% sensitivity for the corresponding serotype and 64% to 90% for other serotypes. Cross-reactivity with Zika, Japanese encephalitis, and West Nile viruses ranged from 21% to 65%, reflecting NS1 antigen conservation. Our study provides valuable insights into the cross-reactivity of DENV NS1 antigens widely used in research and highlights the potential of OMB-based assays for quantitative and epidemiological studies. Ongoing efforts should aim to minimize cross-reactivity while maintaining sensitivity and explore integration with complementary platforms for improved diagnostic precision.

Keywords: Japanese encephalitis virus; West Nile virus; cross-reactivity; dengue virus; non-structural protein 1; optical detection; serology; zika virus.

Figure 1

Optical modulation biosensing principles. (a) An OMB-based serological assay consists of a magnetic bead coated with a capture DENV NS1 antigen, target IgG antibodies, and a fluorescently labeled detection antibody. (b) To aggregate the magnetic beads, a small permanent magnet with a sharp tip is positioned under the sample well. To separate out the background noise of unbound fluorescent molecules, the laser beam is manipulated back and forth from the fixed beads to the background solution.

Figure 2

Clinical evaluation of the OMB-based anti-DENV-1–3 NS1 IgG serological assays. Magnetic beads with a capture antigen (e.g., DENV1-NS1) are mixed with 2 µL of the clinical serum sample and incubated for 60 min. Following a single washing step, fluorescently labeled detection antibody is added and incubated for 30 min. After an additional wash, the plate is loaded into the OMBi system for measurement.

Figure 3

Analytical performance of OMB-based anti-DENV-1–2 NS1 IgG serological assays. (a) Dose–response of a recombinant anti-DENV-1 NS1 IgG antibody using the OMB-based anti-DENV-1 NS1 IgG serological assay, and (b) dose–response of a recombinant anti-DENV-1 NS1 IgG antibody using the OMB-based anti-DENV-2 NS1 IgG serological assay. The limits of detection (LoDs) are 405 ng/L and 3842 ng/L, and the coefficients of variance (CV) are 6% and 10%, respectively. The error bars represent the standard error of three ($n=3$) experiments performed on different days.

Figure 4

Clinical sensitivity, specificity, and cross-reactivity within serotypes of the OMB-based anti-DENV-1–3 NS1 IgG serological assays. Clinical sensitivity, specificity, and cross-reactivity within serotypes of the OMB-based (a) anti-DENV-1, (b) anti-DENV-2, and (c) anti-DENV-3 NS1 IgG serological assays (marked as DENV-1 kit, DENV-2 kit, and DENV-3 kit, respectively). The cutoff for each assay was determined as three standard deviations over the average signal from the healthy patients.

Figure 5

Cross-reactivity analysis of the OMB-based anti-DENV NS1 IgG serological assays with other Flaviviruses and SARS-CoV-2. (a) Cross-reactivity of the OMB-based anti-DENV-1 NS1 IgG serological assay (marked as DENV-1 kit) with WNV, JEV, ZIKV, and SARS-CoV-2. (b) Cross-reactivity of the OMB-based anti-DENV-3 NS1 IgG serological assay (marked as DENV-3 kit) with ZIKV. The cutoff for each assay was determined as three standard deviations over the average signal from the healthy patients.

Figure 6

Non-structural glycoprotein-1 (NS1) sequence similarity among dengue virus serotypes (DENV-1–4), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). The numbers represent the percent identity as calculated by the basic local alignment search tool (BLAST, version 1.4.0, see Supplementary Materials). The color gradient corresponds to percent identity, with dark red indicating high similarity (close to 100%) and light red indicating low similarity (e.g., ~50%).