Development of electrochemiluminescence immunochromatographic test strips for dengue virus

Dengue virus (DENV) infection poses a serious threat to global public health. Rapid and sensitive point-of-care diagnostic methods are urgently needed for early detection and epidemic control.

The dengue virus monoclonal antibody NS1-McAb1# was labeled as the labeled antibody and sprayed onto the binding pad using ruthenium tripyridine as the signal probe, and the dengue virus monoclonal antibody DN-REAB-G2-006 was fixed on the detection pad as the capture antibody. To mitigate potential interference from other substances in the sample, the sample pad and conjugate pad was pretreated with a solution containing heterophilic blocking reagent, and Elecsys buffer was selected as the optimal co-reactant. After dropping the sample onto the sample pad, chromatography and immunoreaction were performed sequentially. Then, cyclic voltammetry scanning was carried out in the test zone at a rate of 0.1 V/s for electrochemiluminescence (ECL) detection. A novel detection method was established by optimizing various parameters.

The ruthenium-labeled antibody was prepared by reacting Ru-NHS ester with NS1-McAb1# at a molar ratio of 50:1 for 10 h. Elecsys Buffer was selected as the co-reactant, with a sample-to-buffer volume ratio of 2:1. Whatman AE 100 membrane was used as the detection pad with a length set to 15 mm. The concentration ratio of labeled antibody to capture antibody was 1:2. The chromatographic time after sample loading was 10 min. Under these optimized conditions, the detection method achieved a minimum detection limit of 20.76 pg/mL, exhibited good specificity, with an intra-assay repeatability coefficient of variation of 4%, a storage stability coefficient of variation of 0.83%, and an inter-assay stability coefficient of variation of 0.81%. Clinical sample validation showed a Kappa value of 0.625 compared with nucleic acid testing results.

The developed ECLIA test strip provides a portable, rapid, and sensitive detection method for DENV, with potential applications in on-site testing across multiple scenarios. Although preliminary clinical evaluation showed acceptable concordance with nucleic acid testing, further large-scale validation using diverse clinical specimens is necessary to fully establish its diagnostic utility in real-world settings.