## 2 Materials and Methods
### Colorimetric Sandwich ELISA for DENV Detection  
Sandwich ELISA, vaunted as the most specific and reliable biorecognition protocol, was selected as the method of interest for minimizing the risk of non-specific bindings and obtaining accurate results [26]. In this protocol, the virus was immobilized between two antibodies (Abs): capture Ab (rabbit anti-dengue virus 2 antibody, ab155042, Abcam, San Francisco, CA, USA) and primary Ab (mouse IgG2a anti-DV, ab155863, Abcam, San Francisco, CA, USA). Since these two Abs are propagated in the bodies of different hosts, they are incapable of binding each other. However, a secondary labeled Ab (anti-mouse IgG2a alkaline phosphatase, ab97242, Abcam, San Francisco, CA, USA) can further bind to the primary Ab and generate the colorimetric detection signal. The chance of cross-reactivity or analytes' interface for the antibodies was reported to be minimal (less than 2%) according to the data sheets received from the manufacturer. Along every single set of the experiments, conventional ELISA in untreated PS 96-well plate (Nunc, Roskilde, Denmark) was also conducted as a control (the sandwich ELISA protocol is described in the supplementary section). To reduce experimental inconsistency, all conducted assays were performed under the same conditions using the same reagents. No major intraday variability was observed for the performed assays in this study. Enveloped dengue virus was prepared in our laboratories via a clinical isolation and processing of dengue serotype 2 obtained from a patient's serum sample (DV2-isolate Malaysia M2, GenBank R Taxonomy No.: 11062).  
Depending on the purpose of the assay, a variety of DENV concentrations was prepared by serial dilutions. In order to calibrate the assay, serial diluted virus solutions in the concentration range of \(3.5\times 10^{-5}\) p.f.u/mL to 3.5 \(\times\)\(10^{-2}\) p.f.u/mL were applied in the assay. Calibration curves were plotted after conversion of the data to the logarithm values. The detection range was studied by running the assay in a wide concentration range between \(3.5\times 10^{-5}\) p.f.u/mL and \(3.5\times 10^{5}\) p.f.u/mL. Detection comparison, however, was selected from the results obtained only for DENV concentration = \(3.5\times 10^{-2}\) p.f.u/mL. Negative controls were calculated from the assays conducted in the absence of virus (\(n\) = 24). Cut-off values for each individual platform were considered as twice of the mean values of the negative controls, which is in accordance with the approved methodologies [15]. Only those replicates that have shown optical density (OD) greater than corresponding cut-off values were interpreted as positives readings. Detection results have been plotted after subtracting the cut-off values from the original data. Positive detection results were accompanied by their negative controls for additional clarification. A total number of 96 samples including 72 positive and 24 negative replicates were examined for each individual platform in sandwich assay to study the sensitivity and specificity of the proposed methodology in contrast to the conventional ELISA. Limit of detection (LoD) for each separate platform was determined from the standard deviations in the case of minimum DENV concentration and slopes of the corresponding calibration plot [26; 27; 28; 29; 30; 31; 32]. Calculations are presented in the supplementary section.