Validation of electrochemiluminescent (ECL)-based immunoassay to determine the levels of antibodies against Escherichia coli O-lipopolysaccharide antigens.

UNLABELLED: Extra-intestinal pathogenic Escherichia coli (ExPEC) is a significant cause of invasive infections, including urinary tract infections, neonatal meningitis, and bacteremia. The emergence of multidrug-resistant ExPEC strains has intensified the need for effective prophylactic interventions. Johnson & Johnson has developed ExPEC10V, a multivalent vaccine targeting ten distinct O-antigens associated with pathogenic ExPEC strains. To assess the humoral immune response elicited by ExPEC10V, an initial enzyme-linked immunosorbent assay (ELISA) targeting E. coli O-polysaccharide (O-LPS) antigens was employed. However, given the vaccine's multivalent antigenic composition, and to improve throughput and efficiency, a multiplex electrochemiluminescent (ECL) immunoassay was developed, qualified, and validated to enable simultaneous determination of IgG antibody titers against all 10 O-antigens, and the carrier protein (EPA) in human serum. The ECL assay was validated for relative accuracy, precision, and linearity using serum samples from an ongoing clinical trial. Precision of incurred samples was systematically assessed across multiple runs to ensure reliable detection of baseline antibody levels. Specificity was validated through competitive inhibition using homologous and heterologous O-LPS antigens. Assay performance was further confirmed by assessing total error, linearity, and dilutional linearity across a broad concentration range. Robustness was tested by varying critical incubation times, while ruggedness was demonstrated using two different ECL plate readers. Sample stability was verified under multiple freeze/thaw cycles and prolonged storage at 4-8°C. The assay demonstrated acceptable precision, relative accuracy, linearity, and robustness across all tested antigens. While most antigens met ruggedness and specificity criteria, O6A failed during simultaneous heterologous competition; however, acceptable specificity was achieved when tested with individual heterologous antigens. To further investigate the O6A outcome, various antigen coating concentrations were evaluated; no improvement in performance was observed at either higher or lower concentrations. Sample handling and storage conditions were comparable to reference conditions, and pre-coated plates remained stable for up to 3 years. Validation of the ECL-based immunoassay for determining IgG antibodies titers against E. coli O-antigens and the EPA protein in human serum was successfully completed, with all parameters meeting predefined acceptance criteria. The assay offers a robust, sensitive, and efficient platform for evaluating vaccine-induced immune responses, supporting both clinical development and future research applications. IMPORTANCE: Extraintestinal pathogenic E. coli (ExPEC) is a major cause of serious infections, and growing antibiotic resistance makes these illnesses harder to treat. Vaccines such as ExPEC10V could help prevent these infections before they start, reducing the need for antibiotics and improving patient outcomes. To understand how well this vaccine stimulates protective antibodies, scientists need reliable tools to measure immune responses. Traditional tests could only evaluate one bacterial target at a time, which was slow and labor‑intensive for a vaccine containing 10 components. By developing a new multiplex electrochemiluminescent assay, we can now measure antibodies to all 10 ExPEC targets simultaneously using a very small amount of sample. This improvement increases efficiency, supports large clinical studies, and accelerates progress toward a preventive vaccine for an urgent global health need.