(M1330-02-09) Qualification of a Novel Multiplex Assay for the Quantitation of Chemokine Markers in Human Plasma and CSF Samples

Purpose: The purpose of this work was to qualify a custom 7-biomarker MSD “U-Plex” multiplex assay for the quantitation of Fractalkine (CX3CL1), GRO-a (CXCL1), IL-18, IL-1RA, IL-8 (CXCL8), SDF-1α (CXCL12), and MCP-1(CCL2) in Human Plasma and CSF samples. Multiplex assays have become an increasingly valuable tool in biomarker analyses. They offer the ability to evaluate multiple targets or analytes simultaneously, while using less sample volume, with decreased time and labor in generating impactful clinical results. Platforms which offer the flexibility of combining multiple biomarkers of interest for a particular disease on the same assay can be even more valuable. Additionally, assays which have been characterized in multiple matrices, such as plasma, serum, CSF, urine, etc. can provide additional value of disease state or drug effect between the various physiological compartments, which may improve understanding of biological interactions and target mediated effects. Here we have optimized and qualified a method to measure 7 individual chemokines, implicated in various autoinflammatory, oncological, and neurodegenerative indications, in both plasma and cerebrospinal fluid (CSF). The successful use of this method may generate useful data for multiple purposes, including characterization of disease state, definition of the relationship between peripheral and CNS, as well as measurement of drug effects.

Methods: Reagents and kit components, including plates, calibrators, and labeled capture and detection antibody reagents were purchased from MSD, (Gaithersburg, MD). Normal and Parkinson disease Plasma and CSF samples were procured from BioIVT, (Westbury, NY). Assay performance assessments included calibrator and sample inter and intra-precision, accuracy, dilutional linearity short term and freeze-thaw stability. Spiked samples in pooled and individual plasma and CSF samples were prepared at multiple concentrations and stored in multiple aliquots at the beginning of the Qualification and stored at -80°C until used for each experiment during the Qualification.

Results: Results are presented in the table below for intra-assay precision, inter-assay precision and accuracy, dilutional linearity, and short-term stability. The target acceptance was based on 25% CV and ±25% RE (30% at LLOQ). Calibrators were common for plasma or CSF, and were within acceptance for precision and accuracy across the entire ranges. Matrix QC’s were prepared at 5 levels spanning the respective calibrator ranges per chemokine, and results are presented for all 5 levels. Results outside of the target acceptance would be considered as fit for purpose based on the context of use of the individual study.

Conclusion: A custom 7-biomarker multiplex assay was qualified in human plasma and CSF. This assay may provide rapid and reliable results to support clinical studies for various autoinflammatory, oncological, and neurodegenerative indications. Additionally, by assessing assay performance in both CSF and plasma, clinical studies may be designed to collect samples from the appropriate CNS or peripheral compartments where biological activity is targeted. This multiplex assay is considered fully qualified for quantitation of 7 chemokines within the concentrations meeting target acceptance criteria and fit for purpose for exploratory use within the full range of calibrators.

References: 1) Paul, D., Basavan, D. Implications of fractalkine on glial function, ablation and glial proteins/receptors/markers—understanding its therapeutic usefulness in neurological settings: a narrative review. Futur J Pharm Sci 8, 56 (2022). https://doi.org/10.1186/s43094-022-00446-0
2) Branch, Donald R. The chemokines IP-10/CXCL10 and IL-8/CXCL8 are potential novel biomarkers of warm autoimmune hemolytic anemia, Blood Adv (2023) 7 (10): 2166–2170. https://doi.org/10.1182/bloodadvances.2021006797