Development and diagnostic evaluation of indirect ELISA for detecting antibodies against the rabies virus glycoprotein in human cerebrospinal fluid.

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.