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The xMAP® technology from Luminex allows for the focused analysis of a defined set of proteins or genes. The PMGC offers services that capitalize on the power of the xMAP® technology using the Bio-Plex™ System from Bio-Rad.

This multi-analyte suspension array incorporates several technologies including fluorescently dyed microspheres (beads), flow cytometry, lasers and associated optics to measure the biochemical reactions that occur on bead surfaces, and a digital signal processor to manage the data. The technology is fast and reproducible with relatively small sample requirements and offers greater flexibility than planar arrays. Up to 100 different biomolecules (proteins, peptides, or nucleic acids) can be analyzed in parallel.

The xMAP® technology is useful for many applications including protein expression (such as cytokines, chemokines and growth factors), protein phosphorylation analysis (signal transduction), HLA testing, and focused gene expression profiling.

                         


Pricing: Download
Please note that this pricing is for academic groups. Commercial customers should contact us for a quote.

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Service Includes:

We offer both a full service option, where trained PMGC technicians carry out the entire xMAP® assay in our laboratory, and a plate-reading service, where clients perform the assay in their laboratory and submit their plates for reading.

The PMGC can process any assay certified for use on the xMAP® platform, including those from Bio-Rad, Millipore, and R&D systems.


Sample Requirements:

Please note that we cannot process infectious human sera.
For cytokine assays Download
For phosphoprotein assays Download

Note: These are the sample preparation guidelines specific for Bio-Rad assays. If you are using an assay from another vendor, please contact us prior to submitting your samples.


How it works:

Assays based on the xMAP® technology use a liquid suspension array with up to 100 uniquely colour-coded bead sets. Each of the 100 bead types are internally labelled with a specific ratio of two fluorophores to assign it a unique spectral address. The beads are then conjugated with different biomolecules (including RNA, DNA, enzyme substrates, receptors, antigens, and antibodies), allowing the capture of specific analytes from the sample. A fluorescently-labelled reporter molecule is then added to the sample in order to detect and quantitate each captured analyte. The beads are drawn through a flow cell where two lasers excite each bead. Fluorescent signals are recorded, translating the signals into data for each bead-based assay. To see a video description of how the Luminex xMAP® technology works please click here.


Quick Questions:

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Where can I find more information about Luminex assay content?
Luminex licenses the rights to create xMAP® assays to a number of xMAP® partners, who in turn create a number of different assays with varying content. As the nature of these assays is to focus on specific subsets of proteins or genes, each assay provider has developed different content, which taken in total gives a fairly broad spectrum of potential analytes to profile.

Since Luminex licences the technology, they are able to keep track of all of the assays that are generated. Luminex has created an excellent tool called xMAP® Kit Finder which can be used to determine if any assays exist for your analyte of interest. Note that if you use this tool, choose the Luminex 100/200 instrument option if you would like PMGC to provide the service for you.
How much sample is required?
The amount of sample required is dependent on the type of assay being performed. The numbers provided here are guidelines only. For more accurate information on sample requirements please contact us.

For Cytokine assays:
  • Plasma: 15 µl
  • Serum: 15 µl
  • Cell culture media: 55 µl

For Phosphoprotein assays:
  • Cell culture or tissue samples should be prepared with the appropriate cell lysis kit (such as the Bio-Rad Cell Lysis kit for Bio-Plex assays).
  • 55 µl of protein lysate in assay buffer should be provided at a concentration of about 400 µg/µl. Please contact us for further guidance.
What is xMAP® technology?

Assays based on the xMAP® technology use a liquid suspension array with up to 100 uniquely colour-coded bead sets. Each of the 100 bead types are internally labelled with a specific ratio of two fluorophores to assign it a unique spectral address. The beads are then conjugated with different biomolecules (including RNA, DNA, enzyme substrates, receptors, antigens, and antibodies), allowing the capture of specific analytes from the sample. A fluorescently-labelled reporter molecule is then added to the sample in order to detect and quantitate each captured analyte. The beads are drawn through a flow cell where two lasers excite each bead. Fluorescent signals are recorded, translating the signals into data for each bead-based assay. To see a video description of how the Luminex xMAP® technology works please click here.

Can the PMGC run xMAP® assays from vendors other than Bio-Rad?

Yes! The Bio-Plex system at the PMGC can also run xMAP assays available from any xMAP® certified vendor including:

Are Luminex assays quantitative?

Some Luminex assays are designed to be used in quantitative manner. For example, most of the cytokine assays, provided by vendors such as Bio-Rad and Millipore, are packaged with a set of standards that can be used to generate a standard curve allowing for quantitation of the individual analytes in a sample. Usually we generate an 8 point standard curve that will have a dynamic range of 3 to 5 logs depending on the individual analyte. Please note, in order to make the assay quantitative, 16 measurements must be made of these standard controls, leaving 80 assays available per plate for your experimental samples.

Other assays such as the phospho-protein assays tend to be relative – whereby the degree of phosphorylation is compared between samples in a ratiometric manner rather than a fully quantitative manner.

What is the difference between a technical replicate and a biological replicate? Which type is most useful, and how many replicates do I need?

A biological replicate involves independent samples (multiple patients, multiple biopsies from an individual patient, etc). Serum or plasma from multiple patients would be profiled independently, or cell culture media from multiple individual cell culture dishes would be assayed. The purpose of a biological replicate is to assess and control for biological diversity.

A technical replicate involves splitting a sample at some point and continuing on with the two aliquots through the rest of the protocol. So for example, a technical replicate might involve taking one serum sample and performing two independent assays from that initial sample. Technical replicates provide an indication of measurement (or technical) error, and are useful for diagnosing problems with the protocol but offer little in the way of statistical power for a biological experiment.

The exact number of replicates required for an experiment is difficult to determine a priori without a proper power analysis. Such a power analysis is not always possible as it requires that you have an estimation of the overall variance, which you often do not have before you perform the experiment. We generally recommend doing as many biological replicates as your budget can accommodate. In general, it is good to have at least three biological replicates per condition. For a more detailed determination of the number of replicates required please contact us as we will be happy to help you design your experiment.

Since many beads are read per sample, are replicates necessary?

The fact that many beads are read per sample, in a single assay is an example of technical replication. This level of “oversampling” ensures that that one particular measurement of a specific analyte in a specific sample is relatively accurate. However, this does nothing to address the biological diversity of a particular response.

While each sample is measured relatively accurately, multiplex protein assays are complex and are much more chemically diverse than a nucleic acid assay such as a microarray. Due to the complexity and occurrence of phenomena such as “matrix effects”, it is often a good idea to measure each sample in duplicate or triplicate (as a technical replicate). Of course, this is dependent on both your budget and the amount of sample you have available.

It is more important to provide a sufficient number of biological replicates (i.e. serum from a large number of patients) in order to provide sufficient statistical power for your experiment.

Is there any data analysis required?

As with any multiplexed assay, there is a level of data analysis that must be performed to help filter noise, perform statistical comparisons, and ultimately to determine the biological meaning of the data. The Bio-Plex Manager v5.0 software provides system control, validation, calibration, data acquisition, and data analysis for multiplex assays. The software will calculate concentration, standard deviation, coefficient of variation, and percent recovery. A standard data normalization is carried out using internal controls to allow for comparison between plates, experiments, and samples. Further data analysis is possible including higher level statistical analysis.

What steps are taken to ensure the least amount of variability?

Multiplexed assays such as Luminex assays are relatively complex, with numerous steps, reagents and instruments involved. Despite this, it has been shown in many studies that the leading cause of variance is the facility that is performing the analysis, followed by the technician that completes the experiment. All of our technicians are highly experienced, and rigorously trained. Each project is assigned a specific technician, who completes the entire project to minimize variability. Furthermore, wherever possible, we use assays and reagents from the same lot. We use the same equipment (plate washer, incubator, plate shaker) throughout the project, all in an effort to ensure the tightest possible data.

Often a project is large enough that it will require multiple days worth of labwork to complete. In these cases we work with the customer to identify which samples are replicates and we split these replicates across the various days to ensure that it is possible to control for any day-to-day variations that may occur.