Evaluation of a Novel Homogeneous Mobility Shift Assay for the Measurement of Human Antibodies-to-Infliximab and Infliximab Levels in Patient Serum

Abstract

Purpose: The list of antibody-based drugs available for the treatment of inflammatory diseases, such as inflammatory bowel disease, is steadily increasing. However, certain patients will generate anti-drug antibodies (ADA) that causes many consequences, including loss of drug efficacy and adverse reactions. Monitoring of patients for drug and ADA levels is not only required by the FDA, but also very important for patient management. Current methods for the assessment of ADA and drug levels include bridge ELISA, radioimmunoassay and SPR, which have many disadvantages such as low drug tolerance in the sample. We have developed a non-radio labeled homogeneous mobility shift assay to measure the antibodies-to-infliximab (ATI) and drug levels in serum from patients treated with infliximab (IFX). This method overcomes many of the limitations of the current methods. Methods: To perform the mobility shift ATI assay, Alexa488 labeled IFX with Alexa488 internal control is incubated with ATI positive serum. After equilibration, the free Alexa488 IFX and bound IFX are resolved by size exclusion HPLC and the intensity of the fluorescence in each peak is measured by a fluorescent detector. The changes in the ratio of the internal control to the free IFX peak are proportional to the amount of ATI. The amount of ATI in the sample is calculated from a standard curve generated with different dilutions of ATI positive serum. Similar methodology and analysis are used to measure the IFX level in the serum. We have performed a full method validation on both ATI and IFX assays, and compared the clinical sample test results with those obtained from ELISA methods. Results: Validation of the mobility shift ATI assay revealed a lower limit of quantitation of 35.4ng/mL in serum, lower than the industry requirement of 250-500ng/mL. The linear range of quantitation is 35.4-790ng/mL. The intra- and inter-assay precision is less than 15% of CV, and the accuracy of the assay is within 20%. IFX drug tolerance in the assay is 100μg/mL in the serum. Sera from 100 healthy subjects were tested to set up the cutoff point of 35.4ng/mL. ATI positive samples analyzed by bridge ELISA from 120 patients were also evaluated by the new method and the results showed a strong correlation between the two methods. However, the new method identified 23 false positive samples from the bridge ELISA. Similar results were obtained from the validation of the mobility shift IFX assay. Conclusion: Results from this study demonstrated the superiority of the mobility shift assay in measuring ATI and IFX in patient serum. This method can also be applied to detect other antibody drugs and ADA in patient serum such as those treated with adalimumab. Disclosure: Shui-Long Wang-Employee: Prometheus Laboratories, Inc. Linda Ohrmund-Employee: Prometheus Laboratories, Inc. Scott Hauenstein-Employee: Prometheus Laboratories, Inc. Jared Salbato-Employee: Prometheus Laboratories, Inc. Rukmini Reddy-Employee: Prometheus Laboratories, Inc. Patrick Monk-Employee: Prometheus Laboratories, Inc. Steven Lockton-Employee: Prometheus Laboratories, Inc. Nicholas Ling-Consultant: Prometheus Laboratories, Inc. Sharat Singh-Employee: Prometheus Laboratories, Inc.