Development and laboratory validation of an electrochemiluminescence ELISA technique for measuring infliximab concentrations and anti-drug antibodies

Abstract

Infliximab is a monoclonal biologic medication used to treat inflammatory and autoimmune disorders. Effective patient management involves quantifying the drug's therapeutic levels and timely detection of anti-drug antibodies (ADA). In this study, we show results of the development and clinical laboratory validation of an electrochemiluminescence immunoassay (ECLIA). The ADA assay employs a bridging ELISA format with biotinylated infliximab as the capture molecule and ruthenium-labeled infliximab as the reporter. For the drug assay, a sandwich format was developed using a biotinylated tumor necrosis factor as the capture molecule and an anti-idiotypic antibody fragment specific to infliximab as the reporter. During assay development, the optimal concentrations for the capture and reporter reagents in both ADA and drug assays were determined. The validation assessed accuracy through two methods: a reporter gene assay (RGA) and sending residual clinical samples to a peer lab utilizing ECLIA methodology. The ADA assay showed a combined 96.1 % qualitative agreement with both RGA and peer ECLIA methods. For the drug assay, comparison with the peer ECLIA method revealed a positive correlation (R² = 0.79) with a Deming Regression slope of 1.02 (95 % CI, 0.86–1.18). Despite also having a positive correlation (R² = 0.82) with the RGA, the drug assay slope was only 0.71 (95 % CI, 0.60–0.83), indicating functional differences between methodologies. Both assays displayed inter- and intra-assay precision below 25 % coefficient of variance. Linearity studies and analytical sensitivity tests indicated the analytical measurement range was 20–1500 ng/mL for the ADA assay and 0.5–40 μg/mL for the drug assay. In conclusion, validation of the infliximab ADA and drug assays demonstrated high sensitivity, precision, and accuracy, meeting criteria for the detection and quantification of infliximab and related antibodies in clinical patient testing.