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specification sheet Human Vascular Endothelial Growth Factor Receptor 2 Simple Plex assay for the detection of human VEGF R2 in serum and plasma (EDTA/Heparin) For research use only. Not for use in diagnostic procedures. Calibration Curve Endogenous Levels The factory generated calibration curve shown below was compiled by averaging 5 replicates of each calibrator from multiple runs. The 5PL curve fit shows calibrator concentration (pg/mL) as a function of signal intensity (relative fluorescent units, RFU). Endogenous levels were calculated from multiple samples. Samples were from apparently healthy volunteers. No medical histories were available for the donors in this study. MEAN (pg/mL) RANGE (pg/mL) STD DEV (pg/mL) Serum (n=5) 11,317 8968-12,737 1512 EDTA plasma (n=5) 11,207 8355-13,394 1906 Heparin Plasma (n=5) 10,842 8246-12,767 1828 SAMPLE TYPE ULOQ Precision Intra-assay Precision: Each control was tested 16 times in one assay. LLOQ Inter-assay Precision: Replicates of each control were tested in multiple assays performed by at least three technicians using two lots of reagents. PARAMETER Limit of Quantitation Data shown represents typical performance results for Lower Limit of Quantitation (LLOQ) and Upper Limit of Quantitation (ULOQ) of VEGF R2. CONC. (pg/mL) LLOQ 54.3 ULOQ 31,900 Limit of Detection The limit of detection (LOD) of human VEGF R2 is 14.9 pg/mL. The LOD was calculated by adding three standard deviations to the mean background signal determined from multiple runs. LOW QC HIGH QC Intra-Mean (pg/mL) 222 10,677 Intra-assay SD 12.6 529 Intra-assay CV (%) 5.6 5.0 Inter-Mean (pg/mL) 193 9248 Inter-assay SD 24.3 944 Inter-assay CV (%) 12.6 10.2 Correlation This assay has been correlated to the Quantikine® ELISA Kit with a slope of 0.9-1.1 and an R2 value greater than 0.9. Recovery Recovery was not necessary as VEGF R2 shows natural linearity data from a neat sample to a 1:16 dilution. Simple Plex Assay: Human Vascular Endothelial Growth Factor Receptor 2 specification sheet Linearity Specificity (continued) Samples containing and/or spiked with high concentrations of human VEGF R2 were serially diluted with Sample Diluent to produce samples within the dynamic range of the assay. The 111, 121,145, and 189 amino acid variants of VEGF-A do not cross-react in this assay, but demonstrate a low level of interference. Recombinant proteins were added at the level indicated in the table to a mid-level VEGF R2 control. The VEGF R2 control value was decreased by the indicated percentage in the Simple Plex assay. SERUM (n=4) EDTA PLASMA (n=4) HEPARIN PLASMA (n=4) DILUTION PARAMETER 1:2 Avg % of Expected 96 102 102 Range (%) 88-104 88-114 95-106 Avg % of Expected 101 105 94 Range (%) 92-109 91-127 89-99 Avg % of Expected 89 99 96 Range (%) 82-97 81-112 84-103 Avg % of Expected 91 97 94 Range (%) 80-101 87-110 86-102 1:4 1:8 1:16 % DECREASE human VEGF-A111 1563 16 human VEGF-A121 3125 15 human VEGF-A145 6250 5 human VEGF-A189 3125 5 Sample Collection and Storage The sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated. Serum: Use a serum separator tube (SST) and allow samples to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles. Specificity This assay recognizes natural and recombinant human VEGF R2. The factors listed were prepared at 50 ng/ mL in Sample Diluent and assayed for cross-reactivity. Preparations of the following factors at 50 ng/mL in a rhVEGF R2 control were assayed for interference. No significant cross-reactivity or interference was observed. Recombinant human: • Angiogenin • Angiopoietin-like 7 • Tie-1 • Angiopoietin-1 • Tie-2 • Angiopoietin-2 • VEGF-A165 • Angiopoietin-4 • VEGF-B167 • Angiopoietin-like 3 • VEGF-C • Angiopoietin-like 4 • VEGF-D • VEGF/PlGF • Angiopoietin-like 5 • VEGF R1 • Angiopoietin-like 6 Toll-free:(888) Toll-free: (888)607-9692 607-9692 Tel: (408) (408)510-5500 510-5500 Fax: (408) Fax: (408)510-5599 510-5599 [email protected] [email protected] proteinsimple.com proteinsimple.com AMOUNT ADDED (pg/mL) RECOMBINANT FACTOR Plasma: Collect plasma using heparin or EDTA as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed or icteric samples are not suitable for use with this assay. Sample Preparation Serum and plasma samples require a minimum 10-fold dilution with sample diluent. Samples above the ULOQ require further dilution. © 2015 ProteinSimple. Ella, Simple Plex and the ProteinSimple logo are trademarks and/or registered trademarks of ProteinSimple. 2 D10-1022-001 / Rev A Rev B.