ProcartaPlex Assays Support—Getting Started

The Luminex assay is a bead-based immunoassay that uses beads of defined spectral properties conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration and test samples) into the wells of a microplate. The target protein binds to the capture antibodies over the course of a 2 hr incubation. After incubation on a shaker, the beads are washed by putting the 96-well plate on a flat magnet for 30 seconds, after which the fluid is discarded by flicking the wells or by using an automated plate washer. The magnet is removed, and the beads are resuspended in the detection antibody. Another incubation and wash are followed by the addition of streptavidin–R-phycoerythrin (SAPE). The beads are then washed and are ready to analyze. The Luminex technology is compatible with the following Luminex analyzers:

• MAGPIX System—affordable, efficient, and compact
• Luminex 200 System—versatile, efficient, and widely used in multiplexing
• FLEXMAP 3D System—high throughput (up to 500 simultaneous assays) and automation compatible

ELISA is a simple and powerful way to quantify individual proteins specifically in complex samples. The selectivity of ELISA is achieved through the use of qualified single- or double-antibody sandwich technology, and accurate quantitation is achieved through the use of calibrated standards. ELISAs can detect low-level proteins and can be performed in a 96-well format with only 60 mins of hands-on time. In addition, the results obtained with ELISAs are generally very reproducible.

While ELISA has been established as a standard method of protein analysis, multiplexing methods that enable the measurement of multiple analytes simultaneously in a single sample address a number of specific limitations:

• ELISA allows for the measurement of only one analyte at a time in a given sample, limiting investigators’ increasing need to measure multiple targets in their research studies.
• The low available volume of many samples being studied may limit the number of times analyses can be conducted. This is especially true in small animal research, in pediatric testing, and in microplate assays providing limited sample volumes. The ability to assay multiple analytes in a single small-volume sample enables more effective use of each sample.
• Difficulties in data interpretation can arise when comparing analyte levels measured by multiple ELISAs, each assay having been performed with different sample aliquots and each susceptible to systematic errors leading to decreased precision and accuracy.
• Many analytes require assays with broad dynamic ranges to avoid repeat testing or out-of-range values. Multiplex assays can be designed to have large dynamic ranges for all of the analytes, or ranges tailored to various expected analyte concentrations.

ProcartaPlex multiplex assays, which are based on Luminex xMAP technology, provide a versatile platform that gives users more flexibility and a greater array of options for analyte detection. Whether you are testing for single or multiple analytes, ProcartaPlex multiplex assays deliver accurate analytical performance using efficient, easy-to-follow protocols. Each of these assays has undergone the same development, validation, manufacturing, and quality control standardization we conduct for our ELISAs. Each lot of ProcartaPlex multiplex assays as well as ELISA assays is fully qualified with the appropriate sample type (i.e., species-specific serum, plasma, and cell culture supernatants), and each lot is evaluated based on the following performance characteristics:

• Specificity—each analyte is screened to make sure there is no significant cross-reactivity with other analytes in the multiplex test
• Sensitivity—each analyte is evaluated for both functional sensitivity (differentiation from background) and lower limit of detection (LLOD)
• Precision/accuracy—multiplex assays have good intra-assay precision (<10% CV), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV); these values are comparable to or better than most ELISA tests

ProcartaPlex multiplex assays are regularly tested against the matching ELISAs. Therefore, you can switch easily from ProcartaPlex assays to ELISA and vice versa with reliable results. Most of our ProcartaPlex assays use the same antibody pairs as our traditional plate-based ELISAs, resulting in high correlation (R2 > 0.9) between the two assays.

Results of an experiment measuring IFN gamma and TNF alpha in stimulated human and mouse peripheral blood mononuclear cells (PBMCs). Supernatants were serially diluted two-fold in normal human or mouse serum, to allow for differential cytokine levels in a serum matrix. Additional cytokines for human and mouse analytes were compared, including IL-1 beta and IL-17A, and similar performance was obtained (data not shown).

Here is an overview of Luminex instruments we offer for multiplex detection and analysis.

The MAGPIX System is a compact benchtop instrument with a multiplex capability of up to 50 analytes. It is a robust and cost-effective multiplexing tool. Streamlined startup and shutdown protocols, and minimal maintenance requirements, make the system easy to use—ideal for both new and experienced users. It features simple out-of-the-box setup and interactive software. The MAGPIX System analyzes magnetic beads immobilized with a magnet, excites the beads using light-emitting diodes (LEDs), and then detects and analyzes the beads using a CCD camera.

Please refer to the assay protocol for kit-specific instructions and your Luminex instrument user guide for instrument-specific instructions. Prior to running the assay, please ensure that the probe height has been calibrated with the 96-Well Flat Bottom Plate supplied with the kit. Failure to adjust the probe height can cause damage to the instrument or low bead count. The Luminex system allows for calibration of low and high RP1 target values. We recommend RP1 low target value settings for immunoassays. Please refer to the lot-specific Certificate of Analysis provided with the kit for bead region and analyte associations when entering the information into the Luminex Acquisition Software.

Please review the following table for instrument setup for 96-well ProcartaPlex assays:

^[1] MAGPIX volume can be changed during the run to optimize bead count.

Each protein detected requires a different bead region to differentiate between analytes. The Luminex 200 Instrument System, Luminex FLEXMAP3D Instrument System and Luminex xMAP INTELLIFLEX System are outfitted with two lasers. The first laser is a red diode laser that classifies beads (region) based on color and hence identifies which protein is being measured. The second laser is a green YAG laser that quantitates reporter fluorophore (R-Phycoerythin, RPE) associated with the bead surface and determines the amount of the protein that is bound to the bead. The beads pass through the system at a rate of several thousand per second, excited first by the red laser and then the green laser. The results obtained show protein identity and the intensity of the signal, respectively. The intensity of the signal is directly proportional to the concentration of the analyte captured in the sample. Therefore, by monitoring the spectral properties of the beads and the amount of associated RPE fluorescence, the concentration of one or more proteins can be determined.

The Luminex beads should be protected from light because they are susceptible to photobleaching. We recommend protecting the beads by keeping containers covered with aluminum foil during all incubation steps, and exercising care during handling. The beads should not be frozen, subjected to excessive heat, or exposed to organic solvents.

“Convenience” multiplex panels are pre-mixed and optimized bead sets for use without modification. “Combinable” multiplex panels are target sets which can be combined or supplemented by addition of beads from ProcartaPlex Simplex kits. These two types of panels cannot be multiplexed together.

Yes, please use our ProcartaPlex Panel Configurator to request a quote for your Mix & Match panel.

When using the ProcartaPlex Panel Configurator, your targets will be listed along with any 'notes' or 'cautions' pertaining to your panel selection and sample type.

A complete list of available targets for our entire ProtcartaPlex assay portfolio can be found on our website by using the ProcartaPlex Panel Configurator and choosing the species of interest. Alternatively, a list of all human ProcartaPlex kits can be pulled up on our website using the search term 'ProcartaPlex Assay Human', or by opening the drop-down menus for Human ProcartaPlex Panels on our ProcartaPlex Assays page.

A complete list of available targets for our entire ProtcartaPlex assay portfolio can be found on our website by using the ProcartaPlex Panel Configurator and choosing the species of interest. Alternatively, this information can be found on our ProcartaPlex Assays page.

When combining a Multiplex kit with Simplex Bead Sets, a Basic Kit is not required. All the non-target specific reagents which are included in the Basic kits are components of our Multiplex kits. Only when you are combining multiple Simplex kits (i.e., no Multiplex kit in the panel) will you need to also purchase a Basic kit.

We have listed all the species we have tested in our Cross-reactivity Chart for NHP ProcartaPlex Simplex Kits. The chart can be found here by clicking on the link for Non-human Primate ProcartaPlex Panels.

No, the Basic Kits are specific to the species listed, i.e., the Mouse Basic Kit can only be used with Mouse Simplex Bead Sets. The Mouse, Human, Canine, Porcine, Rat and NHP ProcartaPlex Basic Kits are not interchangeable.

Yes, you can combine ProcartaPlex Combinable panels and Simplex kits from different orders to run a bigger multiplex experiment. You will want to ensure that there is no overlap of bead regions between the kits you are combining. Please refer to the online ProcartaPlex Panel Configurator.

ProcartaPlex High Sensitivity assays are designed to measure small concentration differences in cell culture supernatants, plasma, and serum samples with 10-fold lower LLOQs (Lower Limit of Quantification) and sensitivities for all analytes in the femtogram range. They have been further optimized to overcome the sensitivity detection limits of conventional multiplex immunoassays.

In-house, we test ProcartaPlex assays with RPMI + 0.5% FCS. You would have to empirically determine if your specific medium will interfere with the assay.

Note: Our customers have successfully used media containing up to 5% FBS with ProcartaPlex assays. As bovine serum contains TGF beta, the use of FBS or FCS should be avoided with TGF beta ProcartaPlex assays.

In general, sensitivity between Simplex and Multiplex ProcartaPlex assay kits is comparable.

We do list many references for our ProcartaPlex portfolio on our website here. More recent publications can be found using internet search tools such as Google Scholar or similar.

The ProcartaPlex kits are guaranteed until the expiration date written on the Certificate of Analysis. All kits are released with a minimum of 12 month shelf-life.

Typical timeline for sample to results for our ProcartaPlex assays is 4.5 hrs.

No. To analyze ProcartaPlex (Luminex) plates, you will need access to a Luminex analyzer such as a Luminex 100/200, MAGPIX, or FLEXMAP 3D system.

Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to clot for 20-30 mins at 20-25°C. Centrifuge at 1,000 x g for 10 mins at 20-25°C and collect the serum fraction. Alternatively, a serum separator tube can be used following the manufacturer's instructions. Use immediately or store aliquots at -80°C. Avoid multiple freeze-thaw cycles.

Collect plasma samples in sodium citrate or EDTA tubes. If using heparin as an anticoagulant, no more than 10 IU of heparin per mL of blood collected should be used to prevent assay interference that can result in a false positive signal. Centrifuge samples at 1,000 × g at 4°C for 10 mins within 30 mins of collection. Collect the plasma fraction and use immediately or store aliquots at –80°C. Avoid multiple freeze-thaw cycles.

Cells should be in log-phase growth. Stimulate cells as desired in appropriate cell culture flasks. Using sterile technique, remove the desired volume of conditioned cell culture medium with a pipette and transfer the medium to clean polypropylene microcentrifuge tubes. Centrifuge the medium at 14,000 rpm for 10 min at 4°C in a refrigerated microcentrifuge to remove any cells or cellular debris. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes. These samples are ready for the assay. Alternatively, clarified medium samples can be aliquoted and stored at –80°C for future analysis. Avoid multiple freeze-thaw cycles. Frozen samples should be allowed to thaw on ice just prior to running the assay. Thawed samples should be clarified by centrifuging at 14,000 rpm for 10 min at 4°C in a refrigerated microcentrifuge prior to analysis to prevent clogging of the Luminex™ probe and/or filter plate. Follow the assay procedure provided with the kit for appropriate dilutions.

Collect spleen, lung, brain, kidney, liver, or heart tissue and treat with or without LPS (100 µg, i.p., for 15 mins, 30 mins, 1 hr, 2 hrs, or 3 hrs). Weigh tissue in a 2 mL microcentrifuge tube. Add 500 µL of Cell Lysis Buffer (Cat. No. EPX-99999-000) per 100 mg of tissue. Add one 5-mm Stainless Steel Bead, then assemble tubes into TissueLyser according to the manufacturer’s recommendations. We recommend using 5-mm Stainless Steel Beads from Qiagen (Cat. No. 69989). Homogenize tissue at 25 Hz for 0.5-3 mins as indicated in the table below. Centrifuge the sample at 16,000 × g for 10 mins at 4°C. Transfer the supernatant to a new microcentrifuge tube. Measure the total protein concentration. Dilute samples to 10 mg protein/mL with 1X PBS. To proceed with ProcartaPlex protocol, add 25 µL of Universal Assay Buffer (Cat. No. EPX-11111-000) to 25 µL of the diluted sample to each sample well.

We have not specifically tested synovial fluid samples in-house and therefore these instructions are only our recommendation. Collect synovial fluid into non-heparinized tubes and spin at 1,000 x g for 10 mins within 30 mins of sample collection. The acellular portion of synovial fluid should be stored at –80°C before subsequent analysis. All samples need to be clarified by centrifugation (14,000 rpm for 10 mins) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute samples 1:1 with Assay Diluent prior to addition to the assay. Reference: Raza K et al. (2005) Arthritis Research &Therapy 7(4): R784–R795.

Centrifuge samples at 1,400 rpm for 10 mins at 4°C to remove particulates. Use immediately or store aliquots at –80°C. Avoid multiple freeze-thaw cycles.

We have not specifically tested bronchoalveolar lavage (BAL) samples in-house and therefore these instructions are only our recommendation. The bronchoalveolar lavage (BAL) should be collected in a sterile syringe and kept on ice until ready to be analyzed. Alternatively, BAL can be aliquoted and frozen in usable sample sizes (such that exposure to freeze-thaw is limited to one time). All samples need to be clarified by centrifugation (14,000 rpm for 10 mins) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute 2-fold with Assay Diluent before applying to the plate.

We have not specifically tested cervical secretion samples in-house and therefore these instructions are only our recommendation. Cervical sponges should be placed on ice immediately upon collection. Samples should be stored at –20°C for up to one week and then stored at –80°C until ready for assay. After thawing, sponges should be weighed and placed into Eppendorf tubes, using forceps cleaned with ethanol after each transfer. Add 200 μL of ice-cold extraction buffer (recipe below) to each tube and incubate overnight at 4°C. The sponges and extraction buffer can then be transferred to microcentrifuge tubes with 0.2 μm cellulose acetate filters and centrifuged at 13,000 rpm for 10 mins at 4°C. The eluate can then be tested for cytokine expression.

Extraction Buffer

50 mM HEPES, pH 7.5 

1 mM Na₃VO₄

150 mM NaCl 

1 mM NaF

1 mM EDTA 

0.1% Tween™ 20

25 mM EGTA

10% glycerol

We have tested breast milk samples in-house using the serum ProcartaPlex protocol and found no general interference with the breast milk matrix. Please note that we have only tested a few analytes (TNFa, IL-10, TNFR), however, there is no obvious reason why other analytes should not work as well.

We have not specifically tested ProcartaPlex assays with tear samples in-house. However, here is a reference from the literature describing the use of ProcartaPlex assays with tear samples.

We have not specifically tested oral mucosal transudate samples in-house and therefore these instructions are only our recommendation. Isolate the site around the tooth and insert a piece of periodontal filter paper into the gum pocket around the tooth for 30 secs. Remove the filter paper and extract 4 times with 50 μL PBS for 5 mins each at room temperature. The individual extractions can be combined and analyzed. Dilute 2-fold with Assay Diluent before applying to the assay.

We have not specifically tested saliva samples in-house and therefore these instructions are only our recommendation. Saliva contains several proteolytic enzymes. It would be important to centrifuge samples and be sure not to pipet any cellular material or debris into the assay plate. We would suggest adding some anti-protease in the sample (for example, trasylol or aprotinine, 10 to 50 U/mL) to protect the protein from enzyme degradation. You may then centrifuge the samples at 1000 x g at 4 degrees C for 10 mins to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.

To process cell lysates (extract cellular proteins), follow the instructions provided here.

We have not tested the use of fixed samples with the ProcartaPlex assay and therefore cannot confirm compatibility at this time.

If you have a magnetic automated plate washer, this can be used with our magnetic bead kits.

Our ProcartaPlex assays have been validated with the Hand-Held Magnetic Plate Washer (Cat. No. EPX-55555-000). We also recommend the Magnetic 96-Well Separator (Cat. No. A14179) as it has a stable magnetic field distributing beads along the bottom of the wells for maximum retention of beads during wash steps.

No, we do not recommend centrifuging the magnetic beads for prolonged periods of time.

For better accuracy, we recommend that you run samples/standards as duplicates at a minimum.

There are two points at which the ProcartaPlex assay can be stopped and continued the next day:

• After the addition of standards and samples, the ProcartaPlex assay plate can be stored on a level surface overnight at 2-8 degrees C, covered, and protected from light. When you are ready to continue the assay the next day, bring the assay plate up to room temp on an orbital shaker (600 rpm), still protected from light, and continue with the assay protocol. Please note that ProcartaPlex Quality Control assay validation is always performed after a 2 hr incubation at room temperature.
• After completing the assay, if the plate cannot be read on the Luminex instrument on the day of the assay, it can be incubated overnight. Shake the plate for 30 mins at room temperature (600 rpm), then cover the plate and store on a level surface in the dark at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate the next day, bring the plate to room temperature on an orbital plate shaker (600 rpm) still protected from light. Perform a wash step using 100 μL of fresh, room temperature Working Wash Solution/Reading Buffer and perform the analysis. We do not recommend storing the ProcartaPlex assay plate longer than 1 day.

Different antigen standard set vials can be reconstituted simultaneously as long as the volume of sample type-specific standard buffer is at least 50 µL per vial and equals 250 µL in total volume. Please refer to our application note: Preparation of Antigen Standards.

The recombinant standards included with the ProcartaPlex assay are one-time-use standards. Once reconstituted for use, we do not recommend storing and reusing this recombinant standard. With each new experiment, we recommend reconstituting a fresh unused vial of recombinant standard protein.

When using serum/plasma samples with this assay, the protocol requires that 25 μL of sample be diluted with 25 μL of Assay Buffer. The top standard point is diluted in the same fashion, 25 μL of recombinant protein with 25 μL of Assay Buffer. As the dilution ratio between samples and standards is the same, we can use the same standard curve range. However, please note that the overall OD values might be a bit lower than in a cell culture supernatant assay.

Please refer to the lot specific Certificate of Analysis document to check the source of the Standard Mix (i.e., Standard Mix A, B or C) included with each kit. When combining Simplex Bead Sets with other Simplex Bead Sets or Multiplex kits, multiple vials of the same premixed standard (Standard Mix A, B or C) may be shipped. If the combination of analytes you want to analyze in your multiplex assay includes two analytes that require the same premixed standard, use only one vial of the premixed standards to prepare the standard curve. If the kits you want to combine include different lots of the same premixed standard, please choose one vial (of any lot) for your multiplex assay.

This information can be found on the lot specific Certificate of Analysis provided with the ProcartaPlex assay.

You will need to prepare a 7-point standard curve as well as a blank.

This may be possible as a custom order. Please reach out to your Sales Representative or to Technical Support at: techsupport@thermofisher.com with your kit number and lot number; they can work with our manufacturing team to determine availability of lot-specific standards.

No, the standalone ProcartaPlex assay buffers are not lot-specific.

The ProcartaPlex Universal Assay Buffer is needed to avoid matrix interference of serum and plasma with ProcartaPlex assay kit components.

The diluted buffer can be stored at 4 degrees C and should be used within 1 month of diluting.

Yes, ProcartaPlex Streptavidin-PE (Cat. No. EPX-SAPE-000) is available as a stand-alone item. We offer most of our ProcartaPlex buffers and reagents as stand-alone items. A complete list of what is currently available can be found on our website here.

No. Storage of the beads at temperatures below 0 degrees C will damage the beads and render them unusable.

Ideally, we recommend that you analyze the plates immediately. If the plate cannot be read on the day of the assay, it can be incubated overnight. Shake the plate for 30 mins at room temperature (600 rpm), then cover the plate and store on a level surface in the dark at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate, bring the plate to room temperature on an orbital plate shaker (600 rpm), still protected from light. Perform a wash step using 100 μL of fresh, room temperature Working Wash Solution/Reading Buffer and perform the analysis. We do not recommend storing the ProcartaPlex assay plate longer than 1 day.

To minimize inter-assay coefficient of variation between two independent assay plates, it is very important that the same operator performs the assays. Also, we recommend that you follow the incubation times exactly as they are stated in the manual in order to get consistent results. Other tips for minimizing inter-assay coefficient of variation include performing a standard curve in duplicate on each plate and using the same kit lot.

The sensitivity information is based on the 2 hr incubation with samples.

The Limit of Quantitation ranges from the lower Limit of Quantitation (LLOQ) to the upper Limit of Quantitation (ULOQ) The LLOQ and ULOQ are the lowest and highest analyte concentration that can be quantified with acceptable accuracy. The algorithms used to create the standard curves will impact the LOQ range. Depending upon the shape of the curve, a 5 parameter logarithmic curve fit (5PL) may yield better accuracy compared to a 4 parameter logarithmic curve fit (4PL).

In contrast, the LOD (limit of detection) or sensitivity is a calculated value determined in several independent assays. It is defined as the analyte concentration resulting in an absorbance significantly higher than the blank (mean of 6 independent assays plus 2 standard deviations).

There is no information about the lowest detectable concentration (lowest limit of quantification, LLOQ) from LOD/sensitivity.

When the MFI value of the standard value is used as an input, the concentration value that is generated can be used to evaluate the standard recovery. The standard recovery is calculated by taking the ratio of this calculated concentration value divided by the expected amount of standard and expressing that as a percentage. An acceptable range of recovery should be between 70-130%. This reflects a bias of 30%.

We recommend that you plot your data using a 4- or 5- parameter curve fit.

We supply the standard protein in a lyophilized form. Lyophilization is a dehydration process that preserves the perishable standard. We have demonstrated that often, the amount of starting protein prior to lyophilization and that of the final product after lyophilization varies (this could be different from analyte to analyte). To ensure that we deliver the most quantitative and reproducible product, we always calibrate the standard after lyophilization and therefore, the concentration range can vary from lot to lot of the standard.

Open the Bio-Plex Manager and click on “File”, then “Document Export Properties”, then “Output CSV Format” and set “Analytes Labels” to “Name (Region)”. Choose the desired file “Destination”, and click “OK”. Click “Document Export”, then “Export”, and import the generated .CSV file into ProcartaPlex Analysis App on Thermo Fisher Connect.

We validate and release our ProcartaPlex assays with a minimum bead count of 50. In our experience, the assays also work with a bead count of 20 though we have not validated them at this bead count.

Please collect a minimum of 50 beads per bead region as stated in the user guide.

The size of the beads in our ProcartaPlex assays is 6.5 µm.

All Luminex instruments come with xPonent data analysis software that can be used. Alternatively, we offer a free and robust data analysis app "ProcartaPlex Analysis App", that can be found on our website here.

Additionally, please refer to our ProcartaPlex Assays Support Center for data analysis tips and tricks.

With everything working smoothly, here is the approximate read time for one plate on different Luminex instruments:

- FLEXMAP 3D Systems and INTELLIFLEX Systems will read 96 well plates in ˜20 min, and 384 well plates in ~75 min.
- Luminex 100/200 Systems will read 96 well plates in ˜40 min.
- MAGPIX System will read 96 well plates in ˜50-60 min.

Please note that these are estimates for the read times of the plates only, and do not include time for calibration or set-up of the instrument.