(M1530-09-51) Validation of IL-1RA and IL-2Rα Biomarker Multiplex ECL Assay to Increase Efficiency and Quality in Supporting Inflammatory Biomarker Studies

Purpose: The capability to measure pathway specific and relevant biomarkers has become increasingly important to help progress the development and approval of pharmaceutical therapeutics. The biological activity, therapeutic effect and safety assessments are necessary measurements during clinical trials and are supported through biomarker measurements. These data are often challenging to generate using conventional methodologies. Western blots, ELISA’s, and other conventional platforms require large volumes of matrices, typically measure single analytes, often have low accuracy and precision, or are unable to measure sufficiently low concentrations of analytes (3,4). Recently, high-sensitivity multiplex panels such as Mesoscale Discovery (MSD) U-Plex have been introduced, allowing for the simultaneous measurement of multiple combinations of analytes with increased sensitivity, accuracy, and precision (5). Biomarkers associated with inflammation response and immune system regulation, including IL-1RA and IL-2Rα have been identified as potential prognostic tools for assessing safety and efficacy endpoints, as well as adding diagnostic value in identifying subjects to appropriately stratify clinical trial inclusion criteria (6). Elevated levels of IL-1RA are seen in various indications including rheumatoid arthritis, diabetes, sepsis, cancer, metabolic diseases, and other autoimmune disorders (7,8,9,10,11,12). IL-2Rα is shed upon immune activation and can be used to monitor immune-mediated diseases (13,14), and therefore this multiplex assay is a great candidate to support clinical trial safety and efficacy endpoints involving these inflammatory pathways.

Methods: U-Plex Biomarker Group 1 (hu) Assay kits were procured from Mesoscale Discovery MSD (Gaithersburg, MD). Normal human serum and plasma were procured from BioIVT (Westbury, NY). The method was conducted according to the manufacturer’s recommendations and validated according to current industry standards and regulatory guidance (1,2). Briefly, capture antibodies are conjugated to ‘linkers’ that are specific to spots on the U-PLEX plate surfaces and then coated to the U-PLEX plate wells. Plates are washed to remove any unbound antibodies. Calibrators, controls, and samples are added to the wells, and any analyte present binds to the capture antibodies. Plates are washed to remove unbound material, and ruthenium (MSD GOLD TM SULFO-TAG) conjugated detection antibody is added to the plate and binds to the captured analyte. Plates are washed and MSD read buffer is added to the plates. Plates are placed into the MSD SQ120 reader where a voltage is applied. Sulfo-tag labels emit an electrochemiluminescent signal (ECL) in proportion to the amount of analyte present from the individual spots within the plate wells. The minimum required volume of sample for one run is 80 µL and one plate can run 34 samples.

Results: The validated calibration curve ranges from 1.3 to 5,190 pg/mL for IL-1RA and from 13.3 to 54,500 for IL-2Rα. For human plasma, the validated ranges are 6.6 to 4,629 pg/mL for IL-1RA and 22.6 to 37,473 pg/mL for IL-2Rα. For human serum, the validated ranges are 8.0 to 2,975 pg/mL for IL-1RA and 45.8 to 34,405 for IL-2Rα. Selectivity was tested in 10 normal human serum and plasma individuals, and all passed acceptance criteria. Dilutional linearity was prepared by spiking 2X the ULOQ concentration into 4 normal human serum and plasma individuals and diluting from 1/1-1/64, all samples measuring within the curve range passed acceptance criteria. Parallelism was run in 3 normal human serum and plasma individuals with high endogenous concentrations of both IL-1RA and IL-2Rα an diluted 1/1-1/32, all samples passed acceptance criteria and showed parallelism. Both analytes in both human serum and plasma were stable up to 24 hours at both room temperature and refrigerated. IL-2Rα in human serum is stable up to 3 freeze/thaws and stable up to 5 freeze/thaws in plasma. IL-1RA in human serum and plasma is stable up to 5 freeze/thaws.

Conclusion: A multianalyte method for the quantitation of IL-1RA and IL-2Rα biomarkers in human serum and plasma was fully validated and meets current industry standards for all parameters evaluated. This method will help improve diagnostic and prognostic efforts in various diseases involving IL-1RA, IL-2Ra pathways, by providing data which supports the trial endpoints. This method can help streamline clinical trials by providing decision makers with high-quality biomarker data which can lead to a more targeted and effective way of treating patients and improving their lives.

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Validation Executive Summary of Results