(M1130-01-03) Custom MSD U-PLEX for the Quantitation of CXCL3, CXCL5, and CXCL10 in Human CSF

Purpose: CXC Motif Chemokine Ligands (CXCL) are important biomarkers for a variety of cancers, inflammatory, autoimmune, and neurological disorders. Three of these chemokine ligands, CXCL3, CXCL5, and CXCL10, are known to play an important role in colorectal cancer where CXCL3 and CXCL5 promote tumor angiogenesis and neutrophil infiltration, while CXCL10 modulates immune cell recruitment. This trio has also been shown to have a role in neuroinflammation in multiple sclerosis (MS); CXCL3 and CXCL5 attract neutrophils and macrophages, while CXCL10 recruits T-cells. Given the role these chemokine ligands play in a variety of disease pathology, it is important to be able to quantitate them in parallel. This study aimed to develop a multiplexed method to measure this trio of analytes that would demonstrate adequate sensitivity, robustness and precision. Due to the ability to test custom analytes together, the MSD U-PLEX system was identified as the potential platform to develop this multiplexed assay. However, while MSD offers calibrator material and capture and detection antibodies for CXCL5 and CXCL10, which can be used on its U-PLEX platform, it does not offer the calibrator and antibodies for CXCL3. Therefore, a custom “home brew” MSD U-PLEX method was developed using the MSD provided materials for CXCL5 and CXCL10 along with calibrator material, capture and detection antibodies for CXCL3 from Abcam (Waltham, MA).

Methods: MSD provides antibodies and calibrator protein for the quantitation of CXCL5 and CXCL10. To quantitate CXCL3 along with CXCL5 and CXCL10 using a custom MSD U-PLEX method, unlabeled capture and detection antibody pair for CXCL3 from Abcam (Waltham, MA) were biotinylated and ruthenylated, respectively. Using a challenge ratio of 20:1, human CXCL3 capture antibody with a starting concentration of 1.027 mg/mL was conjugated with 13.70 nmol of biotin. Similarly, using a challenge ratio of 12, human CXCL3 detection antibody with a starting concentration of 1.006 mg/mL was conjugated with 8.048 nmol of SULFO-TAG. To determine the concentrations of the biotinylated and ruthenylated CXCL3 to be used which would give the best S/N, a “checkerboard” experiment was designed and performed. In this experiment, 0.25, 1.0, 2.0, and 5.0 µg/mL of the biotinylated CXCL3 antibody were tested with 0.25, 1.0, and 2.0 µg/mL of the ruthenylated CXCL3 antibody. The standard curve for CXCL3 was optimized across three different runs using different standard 1 and capture antibody concentrations. Using these conditions along with the MSD recommended procedure for CXCL5 and CXCL10, MRD, dilution linearity, selectivity, and prozone effect were evaluated for the 3 analytes in human CSF. Ten individual CSF samples were screened neat, and at dilutions of 2 and 4 folds. Dilution linearity was performed by spiking 5 individual CSF samples with the calibrator material, and serially diluting them to 2, 4, 8, 16, 32, and 64 -folds. Selectivity was performed by spiking 5 individual CSF samples with the calibrator material such that the final spike concentration for each analyte was about 50 pg/mL. Prozone effect was evaluated by spiking a CSF pool and testing it at a final concentration of 5X and 2X above standard 1 of each analyte and at 4- and 16-fold dilution of standard 1 of each analyte. Signals of the samples were compared to the standard 1 signals of the respective analyte.

Results: CXCL3 capture and detection antibodies were successfully biotinylated and ruthenylated. Based on the results of the “checkerboard” run, the optimal S/N ratio for CXCL3 were observed when 5.0 µg/mL of the biotinylated capture antibody and 2.0 µg/mL of the ruthenylated detection antibodies were used. A starting standard curve concentration of 1333.33 pg/mL for CXCL3 gave the best 4 PL curve fitting with the best S/N in the standard curve optimization runs. Using these standard curve and antibodies’ concentrations, the method developed was shown to be selective for the 3 analytes with %CV between the %recoveries across the 5 individual CSF samples being < 20% for each of the analyte. The method also showed dilution linearity up to 64-fold dilution for each of the analytes. The sensitivity of the assay for CXCL3, CXCL5, and CXCL10 was 0.33 pg/mL, 0.48 pg/mL, and 1.32 pg/mL, respectively. The standard curve points for the three analytes were quantified with acceptable accuracy and precision and the developed assay was robust when performed on different days, using different instruments.

Conclusion: This study shows a successful method development and optimization to simultaneously quantitate CXCL3, CXCL5, and CXCL10 using an MSD U-PLEX method with custom antibody conjugation to work on the MSD platform. The method was optimized for precision, accuracy, and throughput, while minimizing effects of interfering factors. Despite its custom nature, it performs similarly to standard MSD assays. The simultaneous evaluation of these three analytes using this method could prove more accurate and precise and more time and cost effective for research studies around various cancers, inflammatory, autoimmune, and neurological disorders.