Upon treatment with protein therapeutics, a subset of patients will typically develop antibodies against the drug. These anti-drug antibodies can be of concern because they have the potential to alter the drug's therapeutic activity. In the case of relapsing-remitting multiple sclerosis (RRMS) patients receiving recombinant interferon-β (IFN-β), those receiving BETASERON® (IFN-β-1b; E. coli expressed, non-glycosylated, des-Met-1, Cys17Ser recombinant IFN-β) have a higher incidence of IFN-β specific antibodies compared to those receiving AVONEX® (IFN-β-1a; mammalian cell-expressed, natural sequence, glycosylated recombinant IFN-β). The current study reports the development and characterization of ELISAs that detect distinct components of the anti-IFN-β response in patients' sera, and therefore can potentially be used to characterize the composition of the anti-IFN-β antibody response. ELISAs were developed using a constant detecting reagent but a variety of IFN-β-derived test antigens (e.g., native IFN-β, biotinylated IFN-β, IFN-β peptides) and capture methods. Assays were characterized using serum samples from a small number of patients treated with recombinant IFN-β (either BETASERON® or AVONEX®). Assays in which IFN-β was captured via a specific mAb, or in which biotinylated IFN-β was captured via streptavidin, detected serum antibodies that recognize IFN-β in its native structural state. In contrast, assays in which IFN-β was coated directly onto the assay plates detected antibodies that recognize forms of IFN-β possessing a folded structure distinct from the native structure. Certain epitopes present on native IFN-β were not represented in these assays in which the test antigen was directly coated on plastic. Antibodies specific for linear epitopes could be detected using linear peptides as test antigens; the locations of these epitopes were mapped by reference to the X-ray crystal structure of IFN-β-1a. Together, these data show that the mode of antigen presentation employed in IFN-β ELISAs determines which antibody specificities are detected, and can affect whether or not a given serum sample is identified as positive for anti-IFN-β antibodies. As a consequence, screening samples in a single ELISA format presenting IFN-β in a non-native form may lead to underestimation of the incidence of IFN-β treated MS patients that have generated antibodies specific to the native, active form of the drug.