Abstract

Background:
Dengue infection has increased significantly in subtropical and tropical regions in recent decades. As of 2020, 15 countries, including Indonesia, reported increasing cases, with mortality rates of approximately 0.5%–3.5%. Since 2014, the Dengue Vaccine Research Consortium under the Ministry of Health in the Republic of Indonesia has advanced a sharia-compliant dengue vaccine prototype in the form of the premembrane/envelope (prM/E) dengue virus (DENV)-1,-2,-3,-4 recombinant proteins.

Aim:
This study aims to develop and optimize an in-house indirect enzyme-linked immunosorbent assay (ELISA) for detecting IgG against prM/E DENV-3, serving as a preliminary antibody titer assessment for candidate dengue vaccines and potential diagnostic kits.

Methods:
Serum samples from mice immunized with 200 ng purified prM/E DENV-3 or 105 Plaque-forming units (PFU)/ml live DENV-3 or Phosphate-buffered saline (PBS) (negative control) were used. ELISA parameters, including antigen concentration (commercial DENV3), serum dilution, and conjugate antibody dilution, were systematically optimized. Optimal conditions were determined to be 100 ng antigen, 1:200 serum dilution, and 1:5,000 conjugate-Ab dilution.

Results:
Seropositive samples yielded absorbance values exceeding the established cutoff. The statistical analysis showed significant differences between groups (p < 0.001).

Conclusion:
The optimized in-house ELISA reliably detects IgG responses to recombinant prM/E DENV3, supporting its utility in early-stage evaluation of dengue vaccine candidates and in the development of diagnostic tools. This study is a successful stage supporting the development of prM/E DENV-1,-2,-3,-4 as a candidate dengue vaccine.

Key words: In-house ELISA; prM/E DENV-3; Dengue vaccine; Method optimization.