Development of multiplexed immunoassay panels for human growth factors and growth factor receptors: bFGF, sFlt-1, PlGF, VEGF, KDR and c-Kit Free

Growth factors and growth factor receptors are targets for cancer therapy and potential biomarkers to monitor disease progression. Electrochemiluminescence-based multiplexed immunoassay panels were developed for simultaneous measurement of multiple analytes per well in a 96-well format. Panel 1 detects the low-abundance analytes - Basic Fibroblast Growth Factor (bFGF), Placental Growth Factor (PlGF), Vascular Endothelial Growth Factor (VEGF), and soluble VEGF Receptor 1 (sFlt-1 ≡VEGFR1), and was optimized for a 25 ìL sample (serum or EDTA plasma). Panel 2 detects the more abundant analytes - soluble VEGF Receptor 2 (KDR ≡ VEGFR2) and soluble Stem Cell Factor Receptor (c-Kit), and was optimized for 50 μL of a 50-fold diluted sample. The assay format is simple: diluent and sample are added to blocked and washed plates, and after a two-hour incubation with agitation, plates are washed, and detection antibody reagent is added. After a second two-hour incubation, plates are washed and read on a MSD SECTOR™ Imager 6000 instrument (throughput of one plate per minute). The lower and upper limits of the assay ranges in the following table represent the analytical sensitivity and the highest calibrator level, respectively. The linear range extends substantially beyond the highest calibrator level. Intra-plate CVs were approximately 4 - 8%. The assays are sensitive enough to measure these biomarkers in normal samples, and the dynamic range extends well beyond the elevated levels expected in disease states. Each analyte in the multiplexed panels is measured accurately even in the presence of a high abundance of other analytes, as demonstrated in an experiment where an elevated concentration of one analyte at a time was spiked into a normal serum sample. Spike recovery and dilution linearity were in the range of 80% to 120%. In conclusion, multiplexed assays for simultaneous measurement of growth factors and growth factor receptors were successfully developed and validated.