Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta.

Objective: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-beta) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk. Method: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-beta and biotinylated IFN-beta, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirrnation assay: Screen "putative positive" samples are tested in the presence of excess drug (preincubation of sera with 0.3 mu g/mL of soluble IFN-beta) and percentage of inhibition is calculated. Results: The assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-beta in human sera as the positive control. Conclusion: An ultrasensitive ELISA for IFN-beta-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-beta with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.