Assay Performance Characteristics
The assays were validated with biological samples and evaluated according to the following parameters: sensitivity, precision, accuracy, working range, cross-reactivity, and matrix effects. Assay sensitivity, defined as the limit of detection (LOD), was determined by adding two standard deviations to the mean median fluorescence intensity (MFI) of ten zero-standard replicates. With the exception of VEGF-D, all the analytes were detected at <5.7 pg/mL in both a serum-based standard diluent and RPMI cell culture medium. VEGF-D had an LOD of 11.5 pg/mL.
Assay reproducibility evaluates both the intra-assay and inter-assay coefficients of variation (reported as a percentage, %CV). The intra-assay (within-run) %CV assesses the variation among the replicates within the assay, whereas the inter-assay %CV measures the variability across three independent assays.
Intra-assay %CV values were calculated from the MFIs of all three replicates at each standard dilution point from representative assays in both serum-based standard diluent and RPMI matrices. All the analytes exhibited <7.4 %CV in both the serum and RPMI matrices. The inter-assay %CV for multiplex assays was determined from three independent plates using the observed concentrations of six-level spiked controls or the quality controls supplied in the kit. With spiked controls, analytes showed <9.6 %CV in both serum and RPMI. The quality controls exhibited <18.2 %CV in standard diluent.
Assay accuracy (also defined as recovery) was calculated as the percentage of the observed concentration value of an analyte relative to the expected value. This parameter was evaluated using standard concentration points and spiked controls in both multiplex and singleplex configurations and in both serum and RPMI. Overall, the standard recoveries were comparable in both matrices, with the majority of analytes showing recoveries of 80–120% within the assay working range.
Assay working range is defined as the range between the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) wherein an assay is both precise (an intra-assay %CV ≤10) and accurate (80–120% of the standard curve recovery). The ranges of these assays were determined for both serum and RPMI cell culture medium.