Firefly particles

Validation, performance, and benchmarking data for FirePlex® immunoassays

Our team works hard to develop, deliver, and guarantee the highest quality products with scientific rigor.

To ensure accurate and reproducible results, we subject our assays to rigorous validation. We comprehensively validate assay sensitivity and dynamic range, specificity, reproducibility, recovery, and linearity among diverse and complex biological sample types, including plasma, serum, CSF, and cell culture supernatants. We also benchmark our products against other platforms, including ELISA, Luminex® assays, and cytometric bead arrays.

With the hard data to support it, you can choose FirePlex immunoassays with confidence for your research needs from disease profiling and biomarker discovery to drug safety and toxicity screening and beyond. 

To ensure accurate and reproducible results, we subject our assays to rigorous validation in line with ‘fit-for-purpose’ biomarker assay development principles. We comprehensively validate assay sensitivity and dynamic range, specificity, reproducibility, recovery and linearity among diverse and complex biological sample types, including plasma, serum and cell culture supernatants. We also benchmark our products against other platforms, including ELISA, Luminex® assays and cytometric bead arrays.
With the hard data to support it, you can choose FirePlex immunoassays with confidence for your research needs—from disease profiling, to biomarker discovery, to drug safety and toxicity screening and beyond. 

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

Contents

Sensitivity and dynamic range
Linearity and recovery
Specificity in multiplex
Biologically relevant quantification
Reproducibility
Benchmarking

Sensitivity and dynamic range - Detect single digit pg/mL concentrations of analytes

FirePlex immunoassays use a combination of Abcam’s carefully screened recombinant antibody pairs, FirePlex particle technology, and rigorous assay development to accurately quantify proteins across a broad dynamic range with high sensitivity down to single digit pg/mL levels. 

We establish assay sensitivity and dynamic range for each analyte within multiplex panels to reflect more relevant experimental conditions. With the standard one-hour incubation protocol, median assay sensitivity is 0.5 pg/mL (typical range 0.22 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) far superior performance, compared to Luminex® assays (Figure 2)   Sensitivity can be further improved and the standard curve extended with overnight incubation.

​​Figure 1. Representative curves from a larger multiplex experiment showing the two (analyte dependent) extended ranges of 0.610,000 pg/mL in mouse (left) and 1.5 30,000 pg/mL in human (right).

Figure 2. Sensitivity benchmarkingFirePlex cytokine assay sensitivity for 128 human and 82 mouse cytokines was benchmarked against the sensitivity stated on the assay datasheet for standard sensitivity Luminex assays from the four leading suppliers. The bars represent the average dynamic range for 128 human targets or 82 mouse targets, with the white line as average sensitivity.

Linearity and recovery - Assay performance guaranteed across diverse biofluids
To confirm assay performance across the full dynamic range and in different biological sample types, we use native linearity of dilution/parallelism and spike recovery studies (Figure 3).

Figure 3. 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.

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 4).

Figure 4. 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 - Ensuring consistent high-performance between singleplex and 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.

All antibody pairs are extensively tested for cross-reactivity of capture and detector antibodies, as well as for protein interference (Figure 5). For >95% of all possible custom multiplex panels, non-specific signals are either insignificant or <1% of the true signal. Incompatible combinations are flagged during panel design, or replaced with antibody pairs that do not cross-react to ensure high specificity with negligible interactions between pairs.

Figure 5. Cross-reactivity testing.  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. The two tallest bars represent two pools tested against the IP-10 capture particle; both contain Eotaxin assay components, but have no other analyte in common. To date, after testing combinations of 344 total analytes, only 13 incompatible analyte combinations (4 mouse, 9 human) have been identified. Click here to expand the image.

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 6).

Figure 6. Representative examples of standard curve alignment testing in singleplex and multiplex.

Biologically relevant quantification - Confirming clinical disease relevance

Throughout assay development, we test assays and panels in clinically-relevant biological samples to ensure that assay sensitivity and accuracy is maintained in a real-life experimental context. We also confirm that results are consistent with those in the literature and published references ranges (Figure 7).

Figure 7. Concentration of 24 native cytokines in human plasma samples from patients with rheumatoid arthritis vs. healthy controls (see equivalent data for mouse) Consistent with the scientific literature and published reference ranges1,2,3, most cytokines and chemokines measured show elevated levels in RA patients 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. Cytokine concentrations were interpolated from standard curves and corrected for sample dilution. Mean ± SD, rheumatoid arthritis n=8, healthy controls n=>4.

See example of testing data for human bronchial lavage samples.

Reproducibility - Data you can trust

Good reproducibility is essential for reliable results. We routinely perform variability analysis on results from the same sample within each experiment and in different experiments performed over multiple days. 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.

Accelerated and real-time time course stability studies are used to confirm reagent stability. Using antibody pairs composed of recombinant monoclonal antibodies ensures high specificity and affinity is retained between lots (Figure 8).

Figure 8. 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. This experiment is performed three times on different days to determine inter-assay CVs.

Benchmarking - Comparison to other immunoassay technologies

Finally, we examine how FirePlex immunoassays perform against other prominent immunoassays. We find an excellent correlation (R value >.95) between results with FirePlex immunoassays, ELISA, Luminex assays and cytometric bead arrays when tested against human serum samples, as well as PHA stimulated and unstimulated PBMC cell culture supernatants. (Figures 9 to 11)

Figure 9. 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 10. 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 11. 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.


Performance guarantee
Assay reliability is as important to us as it is to you
Stringent quality control testing ensures the highest performance standards for assays and reagents. Abcam’s human and mouse recombinant monoclonal antibodies are paired for excellent lot-to-lot reproducibility and femtogram/mL sensitivity of detection. Accelerated and real-time course stability studies confirm reagent stability. Original and subsequent lots are retained as references for subsequent assay production.

With FirePlex Immunoassays, you can rest assured that your data will live up to the high-quality, high-performance standards you expect from Abcam.


See more about how FirePlex compares to other immunoassay technologies



References

  • Brandt, E. B. & Sivaprasad, U. Th2 Cytokines and Atopic Dermatitis. J Clin Cell Immunol 2, 1–25 (2011). 
  • 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). 
  • Lee, J. W. et al. Fit-for-purpose method development and validation for successful biomarker measurement. in Pharmaceutical Research 23, 312–328 (2006).
  •  McInnes, I. B. & Schett, G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat. Rev. Immunol. 7, 429–442 (2007). 
  • Ohl, K. & Tenbrock, K. Inflammatory cytokines in systemic lupus erythematosus. J. Biomed. Biotechnol. 2011, 432595 (2011). 


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

Luminex® is a trademark of Luminex Corporation, registered in the U.S. and other countries.



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