Multiplex pan-filovirus assay performance and reproducibility across varied geographical and resource settings

ABSTRACT Multiplex bead-based immunoassays (MIAs) are promising tools for simultaneously detecting humoral immunity to multiple targets, potentially playing a crucial role in serosurveillance and vaccine response assessments. However, evaluation of assay performance is paramount prior to widespread use. This study presents a performance evaluation of a pan-filovirus MIA through characterization of the analytical range for the EBOV glycoprotein (GP) target and assessments of assay precision and antigen discrimination. The precision of the MIA was evaluated by comparing the detection of anti-filovirus antibodies at two independent laboratory sites: the University of Hawai’i, Honolulu (UH), and the Institut National de Recherche Biomédicale (INRB) in Kinshasa, Democratic Republic of the Congo (DRC). Forty-six samples from Yambuku, DRC, including Ebola virus Disease (EVD) survivors and close contacts, were tested at both sites. Additionally, 858 samples were tested in DRC before and after vaccination with a prophylactic EVD vaccine, ERVEBO. Results demonstrated low variability between laboratories, with intra-assay and inter-laboratory coefficients of variation below predefined thresholds for all filovirus targets included in the multiplex panel. Analyte correlations between sites were high (r 2 =0.86-0.92). Longitudinal analysis detected increased EBOV GP reactivity following vaccination, while reactivity to non-vaccine filovirus antigens remained stable, consistent with minimal cross-reactivity in a vaccinated cohort. These findings suggest that this pan-filovirus MIA produces reproducible results across distinct laboratory settings and may serve as a useful tool for comparative serologic investigations, serosurveillance, and evaluation of EBOV vaccine-associated antibody responses. IMPORTANCE STATEMENT This study investigates the functionality and intra-laboratory consistency of a novel multiplex bead-based immunoassay with pan-filovirus targets. As part of the evaluation process, the feasibility of using the assay in resource-limited settings was demonstrated in the Democratic Republic of the Congo. This assay holds significant promise as a tool for detecting filovirus-specific antibody responses. By leveraging its multiplex capabilities, it may be used for widespread serosurveillance of high-consequence pathogens, including the pan-filovirus antigens such as Ebolavirus and Marburg virus already incorporated in the assay, as well as other targets of interest.