University of Vermont

College of Medicine

Laboratory for Clinical Biochemistry Research

University of Vermont College of Medicine Laboratory for Clinical Biochemistry Research Luminex Validation Overview

Luminex Validation Overview

For general information about our validation studies, please view Luminex Validation Overview (PDF).

If you have any questions regarding this information, please contact Danielle Parent, (802) 656-8984.

Multiplex Immunoassay Validation

The Instrument

The Bio-Plex array system utilizes Multi-Analyte Profiling (MAP) technology developed by the Luminex Corporation (Austin, TX) and comprises a fully integrated system combining hardware and Bio-Plex Manager Software. The Bio-Plex includes enhancements designed by Bio-Rad to increase the dynamic range of the basic Luminex instrument.

The Technology

“Bio-Plex xMAP technology permits the multiplexing of up to 100 different assays within a single sample The system uses a liquid suspension array of 100 sets of 5.5 micron beads, each internally dyed with different ratios of two spectrally distinct fluorophores to assign it a unique spectral address. Each set of beads can be conjugated with a different capture molecule; the conjugated beads can then be mixed and incubated with sample in a micro plate well to react with specific analytes. Following incubation with a fluorescently labeled reporter molecule the contents of each micro plate well are drawn up into the Bio-Plex array reader, and precision fluidics align the beads in single file through a flow cell where two lasers excite the beads individually. The red classification laser excites the dyes in each bead, identifying its spectral address. The green reporter laser excites the reporter molecule associated with the bead, which allows quantitation of the captured analyte. High-speed digital software record the fluorescent signals simultaneously for each bead, translating the signals into data for each bead-based assay.” [Bulletin 2890 Bio-Rad Laboratories, Inc.]

Advantages
  • As little as 12.5 to 25 ul of serum per multiplex assay.
  • Decreased QC variability by performing simultaneous assays on a single sample.
  • Improved correlated data for multiple analytes to reveal complex relationships and pathways of biomolecules.
  • Increased sample throughput by performing simultaneous assays.
Sample Type Options
  • Cell culture supernates allow a broad array of cytokines to be measured.
  • Serum, citrate, and EDTA plasma validations have been performed, with results indicating that some sample types are preferred depending on the analyte but in many cases all sample types can be used.
  • Kits from the following manufacturers have been validated: R&D Systems, LINCO Research, Bio-Rad, BioSource, and Upstate.

Validation Data by Kit Manufacturer

Please note that all validations that were performed used serum or plasma samples from healthy donors, with the exception of the R&D Systems FMAP TNFa comparison to HS TNFa ELISA, which used citrated samples from a study comparing Type 1 vs. Type 2 diabetes vs. healthy donors, and some studies of reproducibility using LINCO's cytokine multiplex kits, which used EDTA samples from a case/control study of post-menopausal women with and without breast cancer.

Many of the following kits have been validated against the gold standard ELISA's normally run in our laboratory. Please note that analytes from all of these kits can be run as singleplex assays. Analytes from selected LINCO and R&D Systems kits have been validated as singleplex assays. Currently we have links to validation data for 24 cytokines or chemikines located on our webpage Luminex Validation Data by Analyte.

LINCO Research
  • Human Adipokine Panel A (Adiponectin, Resistin, total PAI-1 or Active PAI-1)
  • Human Adipokine Panel B (IL-6, Insulin, Leptin, TNFa, and MCP-1)
  • Human TNFa Singleplex assay (validated with cell culture supernates)
  • Human CVD3 (IL-1b, IL-8, IL-10, IFNg, TNFa, VEGF, NT-proBNP)
  • Human Cytokine 21-plex and varying smaller Cytokine multiplexes
BioSource
  • Multiplex Human IL-6, TNFa, MCP-1
  • Multiplex Human TNFa-R1, TNFa-R2
R&D Systems
  • Multiplex Human TNFa, IL-6, MCP-1
Bio-Rad
  • Multiplex Human IL-6, TNFa
Upstate
  • Multiplex Human IL-6, TNFa, MCP-1

Last modified August 05 2013 11:30 AM