A novel homogeneous Biotin-digoxigenin based assay for the detection of human anti-therapeutic antibodies in autoimmune serum.

Electrochemiluminescence (ECL) assays have been widely used for the detection of anti-therapeutic antibodies (ATAs) against biotherapeutics. With the discontinuation of BioVeris (BV) ECL platform, an alternative technology was needed to replace BV assays to ensure continuous support of multi-year clinical studies. After evaluation of several immunoassay platforms, a novel homogeneous Biotin-digoxigenin (DIG) based bridging ELISA format was selected to develop an anti-rhuMAbX antibody screening assay to test serum samples from rheumatoid arthritis (RA) patients. With a homogeneous overnight sample incubation, the Biotin-DIG ELISA achieved comparable relative sensitivity and free drug tolerance to the previous BV ATA assay for rhuMAbX. To abrogate potential auto-antibody interference in RA sera, various assay conditions were thoroughly evaluated and a horseradish peroxidase (HRP)-conjugated chicken anti-DIG antibody was selected as the detection conjugate. Other potential interferences from serum Biotin, naturally occurring anti-avidin antibodies, and concomitant medications such as digoxin and hydrocortisone, which have similar structures to digoxigenin, were also investigated. Under optimized final assay conditions, the Biotin-DIG assay showed a relative sensitivity of approximately 11 ng/mL using a polyclonal anti-complementarity determining region (CDR) enriched positive control; the assay could detect 500 ng/mL of the positive control in the presence of approximately 27 μg/mL of rhuMAbX in RA serum. In addition, a confirmatory step was optimized for the assay based upon pre-incubating serum samples with an excess of free drug. Overall, the Biotin-DIG assay met the performance requirements for an ATA screening assay and had comparable sensitivity and drug tolerance to the BV assay; therefore this assay was a suitable replacement for the BV assay used for previous clinical studies of rhuMAbX. The Biotin-DIG based assay format can be broadly used as an effective screening platform for the detection of anti-therapeutic antibodies.