Aim: Elevated urine chemokine ligand 10 (CXCL10), also known as IP 10, is a biomarker for early detection of renal allograft rejection. To research an affordable, user-friendly, and quick response test for the evaluating urine CXCL10, we explored the utility of a bead-based CXCL10 product and studied the functional characteristics of this assay.
Methods: CXCL10 present in urine samples or standards bind to CXCL10 capture beads, incubated first with a biotin labeled CXCL10 detection antibody, followed by the secondary streptavidin R-phycoerythrin conjugate (SAPE), and then the plate is read on the LABScan3D™ instrument. The standard curve and CXCL10 concentrations are calculated with 4 parameter logistic (4PL) regression using MyAssay software (www.myassays.com).
Results: The overview of the urine CXCL10 bead-based test is shown in Figure 1A. The assay flow is less than 5 hours and less than 2 hours analysis (Figure 1B). A representative standard curve is shown in Figure 2, with R2 >0.99. The spike recovery was evaluated by spiking 6 levels of recombinant CXCL10 into 6 diluted (1:4) healthy urine samples (3.125 -100 pg/ml). Recoveries were determined in 3 independent experiments with duplicates each sample. The recovery ranges from 72% to 115% with an overall mean recovery of 98%. The limit of detection of CXCL10 was below 6.25 pg/ml with intraassay coefficient variation (CV) < 20%. Although the CV at 3.125pg/ml was over 20%, the absolute difference at 3.125 pg/ml was less than 1 pg/ml. The inter assay CV for internal controls were 10% on average; for the urine control samples with spiked CXCL10 from 6.25-100 pg/ml, the inter assay CV ranges from 2%-16% with an overall mean CV% of 7%.
Conclusion: This noninvasive urine CXCL10 bead-based detection test is a quick, sensitive, early screen test, which may provide a cost-effective method to evaluate urinary CXCL10.
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