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Validation data for multiplex immunoassays with Firefly® particle technology.
To ensure that our multiplex immunoassays for flow cytometers work as you expect for use in repeat and high-throughput studies, we rigorously validate for sensitivity and dynamic range, specificity, reproducibility, recovery and linearity. Testing includes the use of complex biological samples, such as plasma, serum and cell culture supernatants.
Assays use a combination of carefully screened antibody pairs, Firefly® particle technology and rigorous assay development to accurately quantify proteins across a broad dynamic range and down to single digit pg/mL levels. Figure 1 shows a representative selection of human analytes. Alternatively, see sensitivity and dynamic ranges for all human and mouse analytes.
Figure 1. Protein standard curves for five human analytes (see equivalent curves for mouse analytes). Assay dynamic ranges are set at either 1.5–3,300 or 4.6–10,000 pg/mL with typical sensitivities ranging between 0.2–2 pg/mL.
As part of assay development, we test extensively in biological samples to ensure that sensitivity and accuracy is maintained in real-life experimental samples.
Figure 2 shows a 24 analyte human panel tested in rheumatoid arthritis and healthy control plasma samples.
Consistent with the scientific literature and published reference ranges1,2,3, most cytokines and chemokines measured show elevated levels versus healthy controls. TNF alpha, IL-2, IL-5, IL-6, and IL-10 healthy controls are not graphed because they fall below the limit of detection.
Figure 2. Concentration of 24 native cytokines in human plasma samples (see equivalent data for mouse). Cytokine concentrations were interpolated from standard curves and corrected for sample dilution. Mean ± SD, rheumatoid arthritis n=8, healthy controls n=>4.
The same 24 analyte human panel was also tested using cell culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA, Figure 3).
Figure 3. Concentration of 24 native cytokines in PBMC tissue culture supernatant samples (see equivalent data for mouse samples). Concentrations of each cytokine were interpolated from standard curves and corrected for sample dilution. Mean ± SD, n=2. Dark blue bars are stimulated, light blue bars are unstimulated.
In addition to plasma, serum and supernatant samples, we also test our assays on samples such as milk and urine. See example testing data for human bronchial lavage samples.
In multiplex immunoassays, many antibody pairs are used in the same mixture. During assay development, we use a combinatorial screen (Figure 4) to identify the small minority of antibody pairs that interfere with each other and subsequently replace most of these. This method enables us to ensure that our antibody pairs are highly specific with negligible interaction between pairs.
Figure 4. Cross-reactivity test. Antibody pairs and protein standards were tested in combinatorial pools to identify antibody pairs that interact. Over the 2,401 pair combinations screened in this experiment, only one combination (Eotaxin and IP-10) was identified as incompatible for use together in a multiplex panel.
Our multiplex immunoassays are tested for spike recovery (Figure 5) and linearity of dilution (Figure 6) to validate performance in the complex mixture of proteins in complex biological samples.
Figure 5. Percent spike recovery. A representative group of analytes are plotted; see datasheets for percent spike recovery data for individual analytes. Spike recovery tests if the same level of signal is detected for a given concentration of protein standard when spiked either into the assay diluent or the biological sample. 100% recovery indicates that none of the components of the sample are interfering with the assay.
Figure 6: Linearity. Determined for six cytokines in human serum. Protein concentration was plotted as a percentage of the expected value. A representative group of analytes are plotted; see datasheets for linearity data for individual analytes. Linearity of dilution tests for assay interference by checking that a proportionate level of analyte is detected when a sample is diluted.
Good reproducibility is essential for data you can trust. We routinely perform variability analysis on results from the same sample within each experiment and in different experiments performed over multiple days (Figure 7). Our assays achieve typical inter-assay CVs of <15% and intra-assay CVs of <10%.
We also perform reproducibility tests using the same samples across different sites, with R2 values consistently above 0.95, indicating good correlation.
Figure 7. Inter-assay CV for 24 human cytokines. The interpolated biological data was measured across three independent experiments (n=3).
1. McInnes, I.B. & Schett, G. Cytokines in the Pathogenesis of rheumatoid arthritis. Nat. Rev. Immunol. 7, 429–442 (2007).
2. Ohl, K & Tenbrock, K. Inflammatory cytokines in systemic lupus erythematosus. J. Biomed. Biotechnol. 432595 (2011).
3. Brandt, E.B. & Sivaprasad, U. Th2 cytokines and atopic dermatitis. J. Clin. Cell. Immunol. 2, 110 (2011).
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