A-286 Development and basic performance of a dengue virus NS1 detection reagent based on Chemiluminescent Enzyme Immunoassay

Abstract Background Dengue fever is an acute febrile infection caused by the mosquito-borne dengue virus (DENV) and is widespread in tropical regions. There are four DENV serotypes, with serotypes 1–3 detected annually. The nonstructural protein NS1 of DENV is produced in infected cells and secreted extracellularly, making it a useful diagnostic marker for acute dengue fever. However, NS1 sensitivity is reduced in secondary infections owing to interference from antibodies produced during the primary infection. We have developed a sensitive DENV NS1 detection reagent based on Chemiluminescent Enzyme Immunoassay (CLEIA) for the fully-automated LUMIPULSE® G1200 system (FUJIREBIO Inc.). We report here its basic performance. Methods Reagent: This reagent detects DENV NS1 in serum or plasma via a two-step sandwich method based on CLEIA. It requires 50 µL of sample and provides results in approximately 30 min. The measurement value, expressed as the cutoff index (C.O.I.), is calculated as a ratio to the cutoff value from the positive control. A value of less than 1.0 C.O.I is considered negative, while a value of 1.0 C.O.I or higher is considered positive. The following performance parameters were evaluated using this reagent. Sensitivity test: A total of forty-five DENV RT-PCR-positive samples were tested using the reagent to verify concordance with RT-PCR. Specificity: Specificity was evaluated using 1024 samples from healthy individuals. Repeatability: Six replicates of 0.5 ng/mL recombinant NS1 solution for each serotype and pooled samples were measured to assess repeatability. All samples used in this study were obtained in full compliance with ethical and regulatory criteria. Results Sensitivity test: The positive concordance rate for forty-five DENV-positive samples with copy numbers ranging from 2.65×10^3 to 2.65×10^10 was 100%. Some samples were positive for DENV NS1 IgM and/or IgG, yet the new reagent was able to detect NS1 antigen in all samples. Specificity: All healthy control samples tested negative, with C.O.I values less than 0.2. Repeatability: The coefficient of variation for the samples ranged from 0.9 to 1.9%, indicating high repeatability. Conclusion The DENV NS1 detection reagent developed for the fully automated immunoassay system exhibited satisfactory performance. This study confirmed that the reagent was able to detect NS1 with high sensitivity, even in the presence of DENV NS1 IgM and/or IgG antibodies. As a highly sensitive, fully automated assay, this reagent may be useful in the diagnosis of dengue fever.