Homogenous Mobility Shift Assay (HMSA) Overcomes the Limitations of ELISA and ECLIA Assays for Monitoring Infliximab (IFX), Adalimumab (ADA), and Associated Anti-drug Antibodies in Serum

Purpose: Commercial ELISA/ECLIA for monitoring IFX and ADA have many shortcomings, including the inability to detect anti-drug antibodies in the presence of drug (Maini, R., et al. 1998; Baert, F., et al. 2003; Hanauer, S., et al. 2004; Bartelds, G., et al. 2007). For drugs given by SQ injection, this is of particular importance, because drug is usually present in patient serum. Further, secondary LOR caused by anti-drug antibodies is common, warranting an assay that can accurately measure anti-drug antibody levels in the presence of drug. This study compares the analytical capabilities of ELISA/ECLIA to HMSA in detecting drug and anti-drug antibody levels in serum. Methods: ELISA/ECLIA assays were developed and validated for IFX, ADA, antibodies-to-infliximab (ATI), and antibodies-to-adalimumab (ATA). Validation was performed according to industry recommendations (Shankar, G., et al. 2011) and included the following parameters: sensitivity, accuracy, precision, and serum interference (normal serum, hemolyzed, lipemic, icterus, rheumatoid factor, and drug). The values derived for ELISA/ECLIA were compared to published results for HMSA (Wang, S., et al., 2012 and 2013). Results: Precision and accuracy for ELISA/ECLIA drug and anti-drug antibody assays were within acceptable limits at greater than 10 μg/mL of drug or anti-drug antibody (CV< 20%, error <25%). However, CVs and error rates increased significantly at concentrations less than 10.0 μg/mL. Concentrations ≤ 1.5 μg/mL for ATI and ATA were not detectable or not accurately measurable by ELISA/ECLIA, but were measurable by HMSA. Drug concentrations as low as 1.0 μg/mL compromised the accuracy of the ATI/ATA ELISA and ECLIA assays, similar to published work (above). In contrast, ATI/ATA HMSA tolerated up to 60 μg/mL of drug. IFX and ADA ELISA/ECLIA showed high sensitivity in buffer (3.12-31 ng/mL); however, sensitivity in serum was less than in buffer (up to 300X). HMSA showed superior accuracy and precision from 0-10 μg/mL drug in serum. In serum with interfering agents, HMSA was able to accurately measure IFX and ADA drug levels at low concentrations (<5.0 μg/mL), whereas ELISA/ECLIA failed. No significant interference was observed at the physiological level of serum substances in the ATI/ATA HMSA, whereas ELISA/ECLIA showed high error rates (30-233%). Conclusion: ELISA/ECLIA assays for IFX, ATI, and ADA, ATA were developed and validated to meet industry standards. HMSA accurately measures drug levels in both normal serum and serum with interfering agents where ELISA/ECLIA do not. We confirm the results of publications showing that ELISA/ECLIA cannot measure anti-drug antibodies in the presence of drug. HMSA has better sensitivity, precision, and accuracy for drug and anti-drug antibodies. Disclosure - Dr. Egea-Pujol - Employee:Prometheus Laboratories, Inc. Mrs. Reddy - Employee:Prometheus Laboratories, Inc. Ms. Patel - Employee:Prometheus Laboratories, Inc. Mr. Christie - Employee:Prometheus Laboratories, Inc. Mr. Salbato - Employee:Prometheus Laboratories, Inc. Mr. Shah - Employee:Prometheus Laboratories, Inc. Dr. Hauenstein - Employee:Prometheus Laboratories, Inc. Dr. Singh - Employee: Prometheus Laboratories, Inc.