All tags Multiplex Multiplex immunoassays with FirePlex® particles: validation and benchmarks

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Multiplex immunoassays with FirePlex® particles: validation and benchmarks

Validation, performance, and benchmarking data for multiplex immunoassays using our FirePlex particle technology.

Multiplex immunoassays based on FirePlex particle technology feature highly customizable analyte panels that use our recombinant monoclonal antibody pairs for highly sensitive assays with a broad dynamic range.

To ensure accurate and reproducible results, we subject the assays to rigorous validation in line with ‘fit-for-purpose’ biomarker assay development principles1,2. We also check correlation with ELISA, Luminex assays and cytometric bead arrays.

Review our data below, or download our full data sheet for a deeper look.


Sensitivity and dynamic range
Linearity and recovery
Specificity in multiplex
Consistency between singleplex and multiplex
Biologically relevant quantification

Sensitivity and dynamic range

We establish assay sensitivity and dynamic range for each analyte within multiplex panels to reflect a more relevant experimental use. With the standard one-hour incubation protocol, median assay sensitivity is 0.5 pg/mL and standard curves typically span either 1.5–3,333 pg/mL in human or 4.6–10,000 pg/mL in mouse (Figure 1). Sensitivity can be significantly improved and the standard curve extended by using an overnight incubation.

Figure 1. Representative curves from a larger multiplex experiment showing the two (analyte dependent) extended ranges of 0.6 to 10,000 pg/mL in mouse (left) and 1.5 to 30,000 pg/mL in human (right).

Linearity and recovery

To confirm assay performance in different sample types, we use native linearity of dilution/parallelism (Figure 2) and spike recovery studies.

Human and mouse analytes are routinely validated in serum, plasma (EDTA, heparin, citrate), cell culture supernatant, and urine. Human analytes are also routinely validated in saliva, milk (defatted), bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), and synovial fluid.

Figure 2. Linearity of dilution studies in native samples plotted as bar (left) and parallelism (right) charts for sTNF RI. Left: Each sample was tested at a 1:4 dilution and at six further dilutions for serum and plasma and at three further dilutions for other sample types. A two-fold dilution series was used. Analyte concentrations were interpolated from the standard curve. Percentage recovery is relative to the 1:4 dilution. Mean recovery and standard error are plotted. Right: For each sample, analyte concentration in the 1:4 dilution was calculated by interpolation against the standard curve. The concentration of each subsequent dilution was then calculated and plotted, based on the dilution factor, from the concentration of the 1:4 dilution.

Specificity in multiplex

Combinatorial testing confirms that individual FirePlex immunoassays retain specificity in multiplex with other assays and further confirms the specificity of the antibody pair against the analyte of interest.

For >95% of all possible custom multiplex panels, non-specific signals are either insignificant or <1% of the true signal.

Figure 3. Example of combinatorial testing using unique detector antibody/protein standard pools. Over the 88 analytes and 3.8 x 1022 possible custom panels tested, only one combination (Eotaxin and IP-10) was identified as incompatible.

Consistency between singleplex and multiplex

As an additional check for assay specificity in multiplex, we test FirePlex immunoassays as both singleplex and multiplex assays to confirm alignment between the standard curves (Figure 4).

Figure 4. Representative examples of standard curve alignment testing in singleplex and multiplex.

Biologically relevant quantification

Throughout assay development, we test assays and panels in clinically-relevant biological samples (Figure 5) to confirm that results are consistent with those in the literature.

Figure 5. Seven cytokines were quantified in normal serum and serum from patients with rheumatoid arthritis (RA), systemic lupus 3-5.


Consistent results between assays is extremely important, and the typical inter-assay and intra-assay CVs are <15% and <10% respectively (Figure 6).

Figure 6. Mean inter-assay CV% values. To determine intra-assay CVs, a single biological sample is tested at three sample dilutions with four replicates of each dilution


Finally, we examine how FirePlex immunoassays perform against other prominent immunoassays. We find an excellent correlation between results with FirePlex immunoassays, ELISA, Luminex assays and cytometric bead arrays (Figures 7, 8, and 9).

Figure 7. SimpleStep ELISA® kits and a FirePlex immunoassay multiplex panel was used to determine human BCA1, IL-17A, GM-CSF, G-CSF, TARC, IL-10 and RANTES concentrations in supernatant from a PBMC cell culture which had been stimulated with 1.5% PHA-M for 24 hours.

Figure 8. Eleven human cytokines (IFN-gamma, IL-4, IL-1 beta, MCP1, TNF-alpha, IL-10, IL-5, IL-17A, IL-2, IL-6, IL-12p70) in stimulated PBMC cell culture supernatants were analyzed with both Luminex assays (Millipore catalog# HCytoMAG-60K, tested by Boston University Analytical Instrumentation Core) and a FirePlex immunoassay panel.

Figure 9. FirePlex assays and bead-based multiplex assays for flow cytometers were used to determine the concentrations of eleven human cytokines in PHA-stimulated PBMC supernatants.

Sensitive, flexible, convenient

FirePlex immunoassays are developed and validated to enable sensitive and accurate multiplexing across a broad dynamic range. Multiplex panels are fully customizable with a broad choice of analytes, and assays can be analyzed on a flow cytometer, with no specialized equipment required, or samples can be sent to our service center.

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Human analytes  |  Mouse analytes


1.  Lee, J. W. et al. Fit-for-purpose method development and validation for successful biomarker measurement. in Pharmaceutical Research 23, 312–328 (2006).
2.  Jani, D. et al. Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development. AAPS J. 18, 1–14 (2016).
3.  Brandt, E. B. & Sivaprasad, U. Th2 Cytokines and Atopic Dermatitis. J Clin Cell Imunol 2, 1–25 (2011).
4.  Ohl, K. & Tenbrock, K. Inflammatory cytokines in systemic lupus erythematosus. J. Biomed. Biotechnol. 2011, 432595 (2011).
5.  McInnes, I. B. & Schett, G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat. Rev. Immunol. 7, 429–442 (2007).

FirePlex® is a registered trade mark in the United States and is an unregistered trade mark elsewhere.

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