Inflammation is the leading cause of mortality worldwide and is indicated in the following eight major disease areas: autoimmune diseases, cancer, cardiovascular diseases, infectious diseases, diabetic complications, metabolic disorder complications, neurological diseases, and pulmonary diseases. Magnetic bead–based assays are widely used for the quantitation of relevant biomarkers implicated in inflammation. Bio-Rad’s Bio-Plex Pro Human Inflammation Assay measures 37 of the most relevant inflammation biomarkers, including TNF superfamily proteins, Treg cytokines, IFN proteins, and matrix metalloproteinases, in a single multiplex panel. This report provides the performance characteristics of the Bio-Plex Pro Human Inflammation Assay Panel, including assay working range, sensitivity, precision, and linearity of dilution, and illustrates the validation data generated using this panel.
Fig. 1. Standard curves with assay controls and serum samples.
Fig. 2. Linearity of dilution.
Fig. 3. Serum levels of biomarkers in normal and disease groups.
Fig. 4. Comparison of the Bio-Plex Pro Human Inflammation Assays to an equivalent multiplex assay from another vendor.
Rigorous Assay Validation
All Bio-Plex Pro Assays undergo a rigorous evaluation that includes the following parameters. Of these, the ones highlighted in bold are indicative of assay performance (Table 1):
- Specificity (cross-reactivity)
- Accuracy (recovery) in key sample matrices; defined as the percentage of the observed concentration relative to the expected concentration of a known amount of analyte within the assay working range
- Inter- and intra-assay precision; defined as the coefficient of variation (%CV) at concentrations within the assay working range
- Sensitivity (limit of detection, LOD); defined as the concentration of analyte for which the fluorescence intensity signal is two standard deviations above the background signal
- Assay working range; defined as the range of concentrations within which the assay is precise and accurate. Boundaries of the assay working range are defined by the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ)
- Linearity of dilution
- Parallelism and matrix effect
- Performance characteristics in real samples
Table 1. Representative performance characteristics.
The assay working range should encompass the biological range of expression in order to be useful in research. Bio-Plex Pro Assays are developed and optimized to ensure real sample data fall within the quantifiable regions of the assay as demonstrated by comparing the standard curves of assay controls to biological samples (Figure 1).
Linearity of dilution displays the ability of an assay to generate measured values from complex samples by comparing the accuracy over a range of sample dilutions. Bio-Plex Pro Assays are designed and validated with high linearity to return accurate results from complex matrices (Figure 2).
Bio-Plex Pro Assays are tested with samples from multiple sources to ensure target analytes are detected within normal biological expression levels and levels associated with disease (Figure 3).
The Bio-Plex Pro Human Inflammation Assays were compared to overlapping assays from vendor R to highlight more reliable sample detection in serum (Figure 4).
Product Configuration Options
Table 2. Comparison of features for each ordering option.