ICBR Proteomics & Mass Spectrometry (PM) Core is now offering access to the Luminex FLEXMAP 3D® analytical platform for discovering and validation of protein biomarkers across a wide range of diseases. The FLEXMAP 3D® system combines differentially dyed fluorescent microsphere sets with an innovative instrument design to enable simultaneous measurement of the concentration of a large number of different analytes in a rapid multiplexed assay capable of detecting up to 500 unique analytes a single sample. Each bead signature is defined by two fluorescent coded dyes (red and infra-red) and is linked to an analyte-specific capture antibody. Antibody-analyte binding is detected using an analyte-specific, biotin-conjugated antibody and streptavidin PE-conjugated second step detector (Figure 1). Analyte levels are determined on the advanced flow cytometry-based FLEXMAP 3D instrument.
The Luminex platform is capable of simultaneously multiplexing up to 500 unique assays at pg/ml sensitivity in a single sample. In a validation study using human cell extracts and a 41-plex bead kit, 26 cytokine and chemokine biomarkers were successfully quantified with a mean intra-assay CV of less than 10%. (Table 1). The standard curves for each analyte were highly reproducible from run to run (data not shown). Based on a two-sided p-value of ≤0.05, 15 out of 26 cytokine/chemokine biomarkers showed significant differences among the three different cell lines (Figure 2).
If you are interested in learning more about this powerful technology please contact the Proteomics & Mass Spectrometry Core at ICBR-Proteomics@ad.ufl.edu.
Table 1. Intra-assay precision of multiplex assays for 26 biomarkers in human multiple myeloma (1-MM), T-cell acute lymphoblastic leukemia (2-T-ALL), and B-cell acute lymphoblastic leukemia (3-B-ALL) extracts. Intra-assay precision is generated from the mean of the % CV’s from 27 reportable results.
|FGF-2||2.3||4.8||2.00 ± 0.02|
|G-CSF||1.8||15.5||2.68 ± 0.02|
|Flt-3L||2.4||6.6||3.85 ± 0.03|
|GM-CSF||3.1||10.1||3.17 ± 0.02|
|Fractalkine||4.5||9.4||1.97 ± 0.01|
|IFNα2||2.4||13.3||3.19 ± 0.02|
|IFNγ||1.6||12||3.46 ± 0.03|
|GRO||2.1||9.2||3.81 ± 0.04|
|MCP-3||1.6||6.4||6.72 ± 0.02|
|IL-13||2.2||9.2||9.41 ± 0.06|
|IL-15||2.7||8.1||6.21 ± 0.03|
|PDGF-BB||2.1||12.3||4.56 ± 0.04|
|sCD40L||3.7||18.9||2.98 ± 0.01|
|IL-7||1.7||16.1||6.94 ± 0.05|
|IL-8||1.9||3.5||9.15 ± 0.04|
|TNFα||2.6||13||5.62 ± 0.04|
|TNFβ||1.6||11.4||6.40 ± 0.02|
|RANTES||1.9||5||5.87 ± 0.04|
|MIP-1α||1.9||14.5||3.09 ± 0.03|
|IL-12P40||2.8||12.4||5.96 ± 0.06|
|MDC||1.6||7.2||5.43 ± 0.05|
|PDGF-AA||4.3||16.7||6.80 ± 0.07|
|IL-9||2.4||8.4||7.16 ± 0.07|
|IL-2||2.1||6.3||4.91 ± 0.05|
|IL-3||2.1||6.1||4.58 ± 0.05|
|IL-5||2.6||10.8||5.16 ± 0.05|