Development and diagnostic evaluation of indirect ELISA for detecting antibodies against the rabies virus glycoprotein in human cerebrospinal fluid.
TL;DR
Overall, indirect ELISA based on the native RABV G provides sensitive detection of intrathecal antibody responses, but it does not consistently predict virus-neutralizing activity.
Abstract
The accurate detection of intrathecal antibodies against the rabies virus (RABV) is critical for antemortem diagnosis and disease monitoring, particularly when the virus-neutralizing activity is low or delayed. In this study, indirect ELISA assays were developed to detect IgM, IgG, and polyvalent (IgA/IgM/IgG) antibodies against the RABV glycoprotein (RABV G) in cerebrospinal fluid. The RABV G antigen, purified by Lens culinaris lectin affinity chromatography, retained its physicochemical and immunoreactive properties, as confirmed by SDS-PAGE, monoclonal antibody recognition and lectin-binding assays. The analytical validation demonstrated low limits of detection (0.056-0.063), high precision and reproducibility (intra-assay CV ≤ 1.84%), acceptable recovery (101.1-115.6%), linear response (R2 up to 0.989), and minimal matrix effects. Diagnostic performance using a composite reference standard showed moderate agreement for IgM (AUC 0.8150) and IgG (AUC 0.7917), and excellent performance for the polyvalent format (AUC 0.9695), which presented the highest sensitivity (88.6%) and overall agreement (84.8%). RFFIT analysis of ELISA-positive samples demonstrated neutralizing activity in a subset of IgM-positive cases, and a higher proportion of IgG- and polyvalent-positive samples. All RFFIT-positive samples were positive in the polyvalent ELISA format. Serial CSF sampling from one patient indicated that ELISA-detected binding antibodies preceded measurable neutralizing activity. Overall, indirect ELISA based on the native RABV G provides sensitive detection of intrathecal antibody responses, but it does not consistently predict virus-neutralizing activity. These findings support the complementary use of binding and functional assays with molecular diagnostics for a comprehensive antemortem evaluation of rabies infection in the central nervous system. Future studies in larger cohorts are necessary to define the clinical applicability.