Help & FAQs: Luminex Assays

Question 1

Answer

Yes, Luminex Performance assays are correlated to the Quantikine® ELISA kits.

Question 2

Answer

Controls are not offered for Luminex Discovery Assays, but may be formulated by the end user. A general guide may be available upon request. Controls are available for select Luminex High Performance Assays and can be found under the supplemental products tab on the product webpage. Some High Performance Luminex kits include controls and the controls will be listed in the materials provided section on the product datasheet. When included in the kit, controls are not sold separately.

Question 3

Answer

It is recommended that a Calibration test be performed within one week prior to running an assay. A Verification test should be performed prior to data acquisition on the day of running an assay.

Question 4

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No, analytes from separate Luminex Performance Assay panels cannot be combined. The base kits contain the standard curves for only those analytes recommended for that specific panel. All buffers and reagents have been optimized for the analytes within that panel and may not be compatible with analytes represented in other panels.

Question 5

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Citrate plasma is not a validated sample type for Luminex assays. Generally, EDTA and/or Heparin have been validated for use as anti-coagulants. Please check the product insert for specific assay requirements.

Question 6

Answer

It is possible to run less than a full plate and use remaining kit components later. However, it is important to note that the microparticle or biotinylated detection antibody cocktails need to be stored as concentrates, and the diluted cocktails should be prepared fresh at the time of the assay. Lyophilized standards and controls are single use only.

Question 7

Answer

R&D Systems Luminex Discovery and High Performance Assays have been optimized to perform with the sample types listed as validated in the kit manual. Unvalidated sample types may be compatible with the assay, but R&D Systems cannot guarantee reliable performance of an unvalidated sample, and further optimization may be needed. It is recommended to run Spike, Recovery, and Linearity testing to determine if a sample type can be reliably measured in the assay. Please contact Technical Service for a Spike, Recovery, and Linearity protocol.

Question 8

Answer

The difficulties of running multiplex beads in a flow cytometer is the inability to distinguish the different bead sets since they are stained with dyes that are optimally excited with a YAG laser. If a customer wishes to run a single bead population (IL-8 only for example), one would need the ability to discriminate different beads with different analyte capturing abilities. Also, since the bead assays are developed with PE-conjugated antibodies, the argon laser of most flow cytometers will be able to generate a signal from these beads. Keep in mind that this does not utilize the full potential of the Luminex Assay kits.

Question 9

Answer

The best method for washing magnetic beads is use of a magnet. However, magnetic beads can be washed using a vacuum manifold and a filter-bottom plate. Magnetic beads are actually larger than the polystyrene (6.4 vs 5.6 um) so there is little risk for the beads to be pulled through the filter with a standard filter bottom plate (Millipore, Cat# MSBVN1B50). Prior to reading the assay, add an extra shaking at the final re-suspension step to be extra sure the beads are free from the plate.

Question 10

Answer

Running the whole plate at one time is not necessary. If running a partial plate, R&D Systems recommends covering the unused portion since the wells are not in removable strips. That way the unused portion of the plate will not be contaminated. Reusing of wells in the plate is not recommended.

Question 11

Answer

R&D Systems Luminex assays are validated by reading the plate within 90 minutes using a Luminex or Bio-Rad analyzer. If an investigator is not able to read the plate within 90 minutes, it is recommended to resuspend the microparticles with 100 μL of wash buffer in each well and seal the plate with an adhesive foil strip to protect from light. The plate can then be stored overnight at 2-8 degrees C. There may be a loss of signal if the assay plate is stored for an extended period. To continue with the assay, bring the stored plate to room temperature on a horizontal orbital microplate shaker (0.12” orbit) set at 800 ± 50 rpm and perform wash step once using room temperature 1X wash buffer. Following the removal of the wash buffer using a magnetic device, resuspend the microparticles by adding 100 μL of Wash Buffer to each well. Incubate for 2 minutes on the shaker set at 800 ± 50 rpm and read the plate within 90 minutes.

Question 12

Answer

R&D Systems does not add HBT reagents to diluents, but instead formulates specialized diluents that are designed to alleviate false positives to achieve the most accurate results. Our diluents are designed to minimize interfering factors such as binding partners, soluble receptors, HAMA, and rheumatoid factor. With R&D Systems immunoassays, it is not necessary to add heterophilic blocking reagents to the samples.

Question 13

Answer

The cost of R&D Systems Luminex kits can vary depending on how many analytes are plexed together. Pricing information can be found on our Luminex Ordering Tool located here: https://www.rndsystems.com/luminex/analytesIf assistance with pricing is still needed, please contact Customer Care.

Question 14

Answer

Determination of maximum storage time for biological samples is rather complex and requires a discrete study design taking into consideration various factors such as sample types, analyte of interest, and integrity of the storage freezer. Bio-Techne lists general guidelines for sample collection and storage conditions in the Luminex product datasheets. However, sample stability has not been evaluated. It is recommended that investigators carry out a stability evaluation for their specific sample types to determine the maximum storage time and/or examine the literature for published studies.

Question 15

Answer

Typically, about 40 samples can be assayed in duplicate. This will depend on the number of points being evaluated for the standard curve and the inclusion of any controls. The Discovery Luminex Assay is typically assayed with a six-point standard curve and High Performance Assays are typically assayed with a seven-point standard curve.

Question 16

Answer

Our Luminex ordering tool is designed to alert you if any analytes are not able to be multiplexed. If analytes cannot be plexed together this can be due to known interference or the same bead region has been selected. If you are having difficulties determining if the analytes can be plexed together, please contact our Technical Support.

Question 17

Answer

Bio-Techne offers two formats of Luminex bead-based multiplex assays - Discovery and Performance Assays. For Discovery Assays - each standard cocktail is a concentrate upon reconstitution with the volume specified on the CoA. After creating Standard 1 following the table in the product insert, the standard 1 value is the concentration listed on the CoA. For Performance Assays - the standard may be 1X or a concentrate after reconstitution, depending on the specific kit. Refer to the Standard Value Card for the reconstitution volume and assigned values. The Standard 1 value is the concentration listed on the Standard Value Card.

Question 18

Answer

A Luminex High Performance Assay consists of select analytes which have been thoroughly optimized and validated together as a panel. The diluents provided in the High Performance kits and assay conditions have been optimized for best results with the analytes on the same panel. The High Performance Assay is the most precise, reproducible and accurate Luminex tool offered by R&D Systems. Controls are available for the Human Luminex High Performance Assays.The Luminex Discovery Assay allows flexibility in customization by mixing and matching analytes across different panels. Analytes in a Luminex Discovery Assay are tested together when built and a final quality control test is performed prior to release. Since each Luminex Discovery Assay is unique and built to order, additional days are required for order fulfillment. Luminex Discovery Assays are not characterized by the same level of optimization and validation testing as the Luminex High Performance Assays. Controls are not available for Luminex Discovery Assay.

Question 19

Answer

Multiplexing calls for carefully chosen reagents and conditions that allow for accurate and meaningful results. Some analytes may not perform well together due to known or unknown biological incompatibility. In addition, analytes may have overlapping bead region. For analytes with overlapping bead regions, please contact Bio-Techne Technical Support to discuss options for bead region reassignment.

Question 20

Answer

Quantist Analysis Software is a commercial Bio-Techne branded software for advanced and intuitive analysis of xPonent data generated with Luminex systems. Quantist Analysis Software enables users to effortlessly edit analyte panels, plate layouts, or sample details, and to evaluate the quality of standards, controls, and unknowns. The Quantist Analysis Software can also be used to easily adjust standard curve parameters, create customizable reports, and perform inter-assay comparisons.

Question 21

Answer

Environmental stewardship is important to R&D Systems and its employees. R&D Systems is ISO 14001:2015 Certified and as part of this we are continuously reviewing our sustainability practices and "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, and glass.

Question 22

Answer

If expected sample values are unknown, an initial test of serial dilutions is recommended. For example, 1:2, 1:10, and 1:50 are typical starting points used by many laboratories.

Question 23

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R&D Systems does not determine the half-life of cytokines in samples such as serum, plasma, cell culture supernates, or cell-conditioned media. The general recommendation is to use the sample immediately or aliquot into single-use volumes and store the samples frozen. Multiple freeze-thaw cycles should be avoided to prevent protein degradation.

Question 24

Answer

The primary purpose of the recommended sample centrifugation step is to remove particulate debris which may interfere with the assay and negatively impact bead acquisition. Sample debris particles may also cause clogging of the instrument sample probe.

Question 25

Answer

Instructional videos on how to use Discovery and High Performance Assays with Intelliflex, FlexMap 3D, Luminex 100/200, and MagPix can be found in the Bio-Techne Academy.

Question 26

Answer

Due to possible variations in techniques, methodologies, and type of instruments, R&D Systems does not provide ranges for its Luminex High Performance controls and recommends that each laboratory determine its own control range. R&D Systems provides target mean values which end users can use for initial determination of their own acceptability range based on tolerance of variations in each laboratory. It is suggested to use the target mean value +/-30%. Note that R&D Systems does not offer kit controls for Luminex Discovery Assays.

Question 27

Answer

Any instrument that is based on xMAP technology can be utilized with our Luminex Assays. Examples of such instruments are Luminex MAGPIX, Luminex 100/200, Luminex FLEXMAP 3D, Bio-Rad Bio-Plex, and Luminex INTELLIFLEX. Several of these instruments are available for purchase from Bio-Techne along with our Quantist Analysis Software for intuitive Luminex data review: https://www.bio-techne.com/instruments/luminex

Question 28

Answer

The product datasheet describes the protocol and validation of the assay which does not change from lot-to-lot. The standard value card is specific for the lot of product manufactured and contains lot-specific values. Please contact Technical Service (techsupport@bio-techne.com) and provide the kit catalog # and lot # if additional standard value cards are needed.

Question 29

Answer

The feature to split based on dilution enables investigators to configure premix Luminex panels based on sample type (cell culture supernates, serum, and plasma). This keeps analytes requiring the same dilution factor within a single panel, while separating analytes requiring different dilutions. The recommended dilution factor for each analyte may be different for each sample type, and the suggested sample dilutions are built into the Luminex Customization Tool. Note that the suggested dilution factors are based on samples from healthy volunteers.

Question 30

Answer

R&D Systems defines sensitivity as the minimal detectable dose that can be reliably distinguished from the assay background. This is calculated by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. The standard curve range has been validated for reliable and accurate performance across multiple kit lots and operators. The lowest standard concentration is the lowest limit that R&D systems can guarantee for reliable assay measurements. Sample values extrapolated below the lowest standard concentration may not be accurate.

Question 31

Answer

R&D Systems immunoassay standards are calibrated against an internal "Master Calibrator" to ensure consistency of the standard curve and sample values from lot-to-lot. The challenge when comparing immunoassay results from different vendors is that there is no single Master Calibrator for each analyte that is used by all vendors. Calibration, coupled with other variables (such as antibodies, detection methods, buffers, and cross-reactivity/interference) can lead to differences in sample values from different vendors’ assays.

Question 32

Answer

Controls are not offered for Luminex Discovery Assays, but may be formulated by the end user. A general guide may be available upon request. Controls are available for select Luminex High Performance Assays and can be found under the supplemental products tab on the product webpage. Some High Performance Luminex kits include controls and the controls will be listed in the materials provided section on the product datasheet. When included in the kit, controls are not sold separately.

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