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ProcartaPlex® Multiplex Immunoassays Frequently Asked Questions


General Questions

Q. What is the shelf-life of the ProcartaPlex kits?

The ProcartaPlex kits are guaranteed until the expiration date written on the Certificate of Analysis.  The kits will have a minimum 6 month shelf-life.

Q. I accidentally stored the whole kit at -20°C. Can I use it anyway?

All the components included in the ProcartaPlex kit are viable except the bead mixture. Storage of the beads at temperatures below 0oC will damage the beads and render them unusable.

Q. Do I need to purchase a Basic Kit when combining Simplex Kits with a Multiplex Kit?

No, when combining a Multiplex kit with Simplex Bead Sets, the Basic Kit is not required. All the non target specific reagents which are included in the Basic kit 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 the Basic Kit.

Q. Can I use the ProcartaPlex Mouse Basic Kit with ProcartaPlex Human Simplex and vice versa?

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.

Q. Can I combine ProcartaPlex kits, which were not purchased on the same order? For example: I purchased a kit 3 months ago and another kit today. Can I combine them or do the kits have to be shipped on the same order?

Yes, you can combine ProcartaPlex 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.

Q. Is it possible to measure Luminex beads on a flow cytometer?

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

Q. How many beads do you recommend we collect? And how do we setup the Luminex machine?

Please collect 50-100 beads per bead region as stated in the product manual.

Q. Can a finished ProcartaPlex plate be read multiple times on the Luminex instrument without a significant signal loss?

Yes, it is possible to reread a finished ProcartaPlex plate without a loss in the signal or the number of beads counted. Please note that the Luminex instrument adds additional liquid to the wells with each analysis.  It is possible that the wells may become overfilled with fluid after the third analysis. Hence, we do not recommend reading the ProcartaPlex plates more than two times.

Q. I have completed the ProcartaPlex assay but can’t run my samples immediately. How long can I store the ProcartaPlex plate before analysis on a Luminex instrument without a significant loss of signal?

Ideally, we recommend that you analyze the samples immediately. If you do need to store your samples, overnight storage at 2-8°C shows no significant signal loss. We do not recommend storing the ProcartaPlex plate longer than 1 day.

Q. I purchased several ProcartaPlex Simplex Bead Sets for use with the Basic Kit and I am preparing the bead mixture.  After I add one bead mix to the plate, I insert the plate into the Hand-Held Magnetic Plate Washer  and invert to remove the bead storage buffer. Can I add the second bead mix to the well while the plate is still inserted into the Hand-Held Magnetic Plate Washer?  Or should the plate be removed from the Hand-Held Magnetic Plate Washer prior to adding the second (and remaining) bead mixes?

The second (and subsequent) bead mixtures can be added to the wells of the plate while still inserted into the Hand-Held Magnetic Plate Washer.

Q. What type of analysis do you recommend for plotting the standard curve?

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

Q. Does the labeling/marking of the plate have any influence on the measurement?

Waterproof markers that dry quickly will not affect the assay. However, markers that do not dry quickly might bleed into the wells during pipetting/washing which could influence the final readout.

Q. What does "Limit of Quantitation" and “Limit of Detection/Sensitivity” mean?

The Limit of Quantitation ranges from the lower Limit of Quantitation (LLOQ) to the upper Llimit of Quantitation (LLOQ) 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.

Q. How are the standard recovery and bias values calculated?

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%.

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Protocol Questions

Q. Can I use a centrifuge for the washing steps in the assay?

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

Q. Can I store reconstituted standard protein for later usage?

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.

Q. Where can I find the stock concentration of the recombinant standards?

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

Q. How many standard points do we need to prepare when using the ProcartaPlex kits?

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

Q. I have bought several Simplex Bead Sets. Which premixed standard do I have to use in my assay?

Please refer to the Certificate of Analysis document to check the source of the Standard Mix (i.e. Standard Mix A, B or C) included with each kit. This is critical to avoid that the same premixed standard protein is added twice to the prepared standard mix.
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 (Standard Mix A, B or C), 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.

Q. When preparing standards/samples, can I run them as single points?

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

Q. I have to cut short the assay and can’t complete the assay.  At which step can we incubate the plates overnight with affecting the assay?

As stated in the product manual, after the addition of the standards and samples, the plate can be stored overnight (Step 11.1.5 or 11.2.5).

Q. I have completed the ProcartaPlex assay but can’t run my samples immediately. How long can I store the ProcartaPlex plate before analysis on a Luminex instrument without a significant loss of signal?

Ideally, we recommend that you analyze the samples immediately. If you do need to store your samples, overnight storage at 2-8°C shows no significant signal loss. We do not recommend storing the ProcartaPlex plate longer than 1 day.

Q. I have stored the finished assay overnight and I want to run it the next day. Do I have to shake the plate again before data acquisition?

Yes, we recommend shaking the plate for 5 minutes before running the plate on the Luminex instrument to ensure homogeneous resuspension of the beads.

Q. If a sample is fixed with paraformaldehyde and processed to separate the serum or plasma fraction after fixation, are these samples compatible with ProcartaPlex?

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

Q. I purchased several ProcartaPlex Simplex Bead Sets for use with the Basic Kit and I am preparing the bead mixture. After I add one bead mix to the plate, I insert the plate into the Hand-Held Magnetic Plate Washer and invert to remove the bead storage buffer. Can I add the second bead mix to the well while the plate is still inserted into the Hand-Held Magnetic Plate Washer? Or should the plate be removed from the Hand-Held Magnetic Plate Washer prior to adding the second (and remaining) bead mixes?

The second (and subsequent) bead mixtures can be added to the wells of the plate while still inserted into the Hand-Held Magnetic Plate Washer.

Q. In preparing the standard curve for serum samples and cell culture supernatant, I will use 25ul and 50ul of recombinant standard to generate the top standard point, respectively.  With the difference in volumes used, how is it possible that it is still the same standard curve for each protocol?

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.


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