BioPlex Bead Based Assays
Multiplex assays of biological fluids using Luminex 100 system
Luminex 100 IS system is developed based on xMAP® technology and it
is a flexible analyzer based on the principles of flow cytometry. The
system enables to multiplex (simultaneously measure) up to 100 analytes
in a single microplate well, using very small sample volumes. This
gives opportunities for performing quick and cost-effective bioassays,
while ensuring optimum accuracy.
The Luminex 100 IS System is the combination of three core xMAP
technologies. The first is xMAP microspheres, a family of 100
fluorescently dyed 5.6 micron-sized polystyrene microspheres that act
as both the identifier and the solid surface to build the assay. The
second is a flow cytometry-based instrument, the Luminex 100, which
integrates key xMAP detection components such as lasers, optics,
advanced fluidics and high-speed digital signal processors. The lasers
excite the internal dyes that identify each microsphere particle, and
also any reporter dye captured during the assay. The third component is
the IS 2.3 software, which is designed for template-based data
acquisition with robust data regression analysis. APAF currently offers
cytokine assay services to customers (single to multiple cytokines) to
be performed from serum/plasma samples (both humans and rodents) using
the Luminex 100 IS system. Cost will depend on number of cytokines in
each sample to be assayed. Contact us for estimated cost prior to
sending samples to APAF.
Key benefits of multiplex assay
- Multiplexing up to 100 bioassays per well (per sample); internal controls allowable in each well.
- Using 50 µL of sample obtains up to 100 results.
- Quantitative results over a 3 to 4 log dynamic range, with strong concordance to ELISA and Mass Spectrometry.
- All
the liquid handling operations for cytokine assays performed on a
robotic motion system for precise and reproducible liquid delivery in
an automatic fashion.
Sample Preparation Guidelines For Multiplex Analysis Using Bio-Plex/Luminex System
Using the Bio-Plex/Luminex system multiplex analysis can be performed for the following molecules/assays:
Cytokines, chemokines and growth factors (human and rodents); signal transduction assays; phosphoproteins; some human disease research assays (acute phase proteins, angiogenesis, diabetes, immunoglobulin isotyping, etc.); some markers for plant diseases; any other protein/bimolecule markers that uses bead based immuno-detection techniques; etc.
APAF conducts all the multiplex assays on a robotic EpMotion liquid handling workstation and reads the assay plate on the Bio-Plex/Luminex system. APAF also has a magnetic bead washer for multiplex analysis for magnetic bead based kits.
Sample Preparation
For serum/plasma or other body fluids and cell culture samples:
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Add protease inhibitor cocktail into the samples in an appropriate concentration.
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Centrifuge samples at 20,000 g for 5 min; discard pellets (bottom) and lipid (top) from the samples particularly for plasma/serum samples. Use middle part of the sample for multiplex assays. For cell culture samples, use the supernatant.
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Filter through a 0.22 μm spin filter for both body fluid and cell culture samples. This is very important for Bio-Plex to run smoothly and having no error in the results. APAF can provide spin filters to customers with an additional cost.
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Ship samples (100 -200 μL/sample) to APAF on dry ice. It requires 75 μL per replicate if the Automatic Robotic Liquid Handling Workstation is used for liquid delivery (50 μL per replicate is required if the assay performed manually).
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For cell culture supernatant samples, approximately 5 mL of cell culture medium itself will have to send to APAF together with the samples. This is required for preparation of standards.
Co-efficient of Variation (CV) of technical replicates is low in the bead based immunoassays. Assays in duplicate or triplicate would be sufficient. However, increasing biological replicates would give better statistical significance.
How do I send sample from overseas to APAF?
If you are sending any biological samples to APAF from countries outside of Australia go to the Quarantine Guidelines in our website or contact us in regards to permits needed for Australian quarantine regulations and the recommended couriers to use.
Pathogenicity
Biological samples coming into APAF should be accompanied by documentation of potential pathogenicity or pathogen free status otherwise APAF will presume all samples from human and animal origin are potential pathogens and will be treated accordingly.
Multiplex Analysis Using Bio-Plex/Luminex System Service Request Form
Once you have followed all the guidelines you can download the
Multiplex Analysis Using Bio-Plex/Luminex System Service Request Form from the website. Fill this form and send to APAF together with the samples.