Step-by-step guide to EYRAplex
Published: April 11, 2025
Updated: April 23, 2025
Authored by: Tyler Sandberg
EYRAplex is Mabtech’s magnetic bead-based multiplex assay designed for sensitive, interference-resistant analyte detection in serum, plasma, and cell culture supernatants. Below you’ll find a full step-by-step guide on how to perform the assay and prepare for analysis using Mabtech EYRA™ or a flow cytometer.
What is EYRAplex?
EYRAplex is a brand-new magnetic bead-based multiplex assay. Combining Mabtech's trusted monoclonal antibody pairs with magnetic beads, EYRAplex assays are designed to deliver reliable results every time. Perfect for measuring >30 analytes in a single sample, saving you time, resources, and precious samples.
The same, highly specific mAb pairs used in our ELISpot, FluoroSpot, and ELISA assays are now available in a new multiplex format with EYRAplex. And with our recombinant mAbs and novel assay buffer, heterophilic antibody interference is a thing of the past. Keep reading to learn more!
EYRAplex kits can be used with either Mabtech EYRA™ or most flow cytometers.
1. Prepare your materials
Make sure you have everything ready before starting the assay. Always consult your kit's datasheet for assay-specific requirements.
You’ll need:
- Reagents and consumables:
- EYRAplex kit
- Capture bead mix*
- Detection mAb mix
- Standard mix
- Streptavidin-PE*
- Streptavidin-PE diluent
- Assay diluent: EYRAplex**
- Wash buffer concentrate
- 96-well black plate with lid and adhesive covers
- Precision pipettes and tips
- Distilled or deionized water
- EYRAplex kit
- Equipment:
- Orbital shaker (suggested 3 mm orbit, 800 rpm)
- Automated or manual 96-well plate washer for magnetic beads
- Mabtech EYRA™ instrument
- Demagnetizer (included with Mabtech EYRA™)
*Note: Protect all fluorescent reagents and the plate from light during incubation steps.
**Note: Store Assay diluent in the fridge in between incubation steps and when not in use.
Washing steps
As with most immunoassays, washing steps are critical for success. We've evaluated several common automated and manual magnetic plate washers and want to share our experience and best practices with them. We also include excess Wash buffer concentrate in each kit to ensure flexibility in your washing protocols whether it's automated or manual.
Automatic washers
BioTek 50 TS with Flat Magnet for 96-well plates (Flat magnet from Agilent 7103016)
- Plate format: Plate
- Prime before washing: Volume 5, Flow rate 5
- Start with one Soak cycle for 90 seconds without shaking
- Four wash cycles:
- Aspiration: Travel rate: 6, Delay: 0, Z-offset: 38, Y-offset: -20
- Dispensing: Volume: 200 µl, Flow rate: 7, Z-offset: 120, Y-offset: 0
- Soak: 45 seconds without shaking
- Final aspiration: Travel rate: 6, Delay: 0, Z-offset: 35, Y-offset: -20
- Remove the plate immediately after final wash
Tecan HydroSpeed with Smart-2 MBS 96 carrier
- Wash head: Type 96
- Plate type: Greiner 96 Flat Transparent
- Aspiration rate 2 with prime before washing
- Start with one Soak cycle for 30 seconds without shaking
- Four wash cycles:
- Aspiration: Mode: Normal, Z-position: Custom 4.6 mm, Time: 2 seconds, Head speed: 10 m/second
- Dispensing: Z-position: Bottom, Volume: 200 µl, Dispense rate: 350 µl/second
- Soak: 10 seconds, no shaking
- Final aspiration: Normal, Z-position: Custom 4.6 mm, Time: 2 seconds, Head speed: 10 m/second
- Remove the plate immediately after final wash
Manual washers
There are several manual magnetic plate washers in 96-well plate formats, and most should work. These two happened to be our favorites.
VP 771HH-F-4 or LifeSep 96F
Regardless of the hand-held washer, for each wash step in the protocol:
- Properly secure the plate on the magnetic rack and let the beads settle for 60 seconds
- Decant the plate while attached to the magnetic rack and gently tap on clean paper towels
- Add 200 µl of diluted wash buffer and soak for 30 seconds
- Repeat for a total of 4 washes
- Remove the plate from the magnetic rack immediately after the final wash
2. Reconstitute and prepare reagents
- Standards: Reconstitute as instructed in the datasheet immediately before use. Standards should be reconstituted fresh for each assay run.
- Wash buffer: Dilute 1:20 (e.g., 50 ml buffer + 950 ml water).
- Detection mAb mix: Dilute in Assay diluent, as instructed in the datasheet, within 15 minutes of use.
- Streptavidin-PE: Dilute 1:100 in Streptavidin-PE diluent just before use and protect from light.
- Samples: Centrifuge thawed samples and dilute if necessary.
- Plasma/serum recommended dilution is at least 1:4 in Assay diluent.
- Cell culture supernatants may be analyzed undiluted or diluted in Assay diluent if expected analyte levels are outside of ULOQ.
3. Add capture beads
The Capture beads mix included with each EYRAplex kit contains a pre-mixed panel of mAb-conjugated beads at a ready-to-use dilution. It's critical to vortex often when handling the beads and protect them from light.
- Vortex the Capture bead mix for at least 30 seconds and immediately add 50 µl per well.
- Beads settle quickly so vortex regularly (either every 30 seconds or every 8-12 wells) while dispensing across the plate.
- Wash 4 times with 200 µl diluted wash buffer per well as described above. Beads will be almost dry after the wash step.
4. Add samples and standards
Standard reconstitution and sample dilution should be prepared on the same day as running the assay. The final volume of standard and sample added per well will be 50 µl so calculate how much to prepare according to your plate layout and use of replicates. We recommend a minimum of duplicates.
- Add 50 µl of diluted sample or standard per well.
- Cover with adhesive film and black lid.
- Incubate 2 hours at room temp or overnight at +4-8 °C on an orbital shaker (800 rpm).
- Wash 4 times with 200 µl diluted wash buffer per well as described above.
5. Add Detection mAb mix
Just before washing the plate after sample/standard incubation, prepare the Detection mAb mix by diluting in the Assay diluent. Once again, 50 µl of diluted mix will be added per well so calculate accordingly.
- Add 50 µl per well of the diluted Detection mAb mix.
- Cover and incubate 1 hour at room temp, on an orbital shaker at 800 rpm.
- Wash 4 times with 200 µl diluted wash buffer per well as described above.
6. Add Streptavidin-PE
The Streptavidin-PE needs to be diluted in the Streptavidin-PE diluent. This should also be prepared just before the previous wash step. Again prepare for 50 µl per well.
- Add 50 µl per well of the diluted Detection mAb mix.
- Cover and incubate 30 minutes at room temp, on an orbital shaker at 800 rpm.
- Wash 4 times with 200 µl diluted wash buffer per well as described above.
7. Prepare for analysis with Mabtech EYRA™
Congrats! The final wash step is finished! Mabtech EYRA™ has been optimized for analyzing EYRAplex wells with 50 µl of Assay diluent.
- Add 50 µl per well of Assay diluent.
- Demagnetize the beads with the demagnetizer.
- Switch on the demagnetizer and swipe the plate across the surface. Rotate the plate 90° and swipe again.
- Let the beads settle for 20 minutes before data acquisition with Mabtech EYRA™.
- Plates can be read right away or stored in the fridge overnight for reading the following day for convenience.
7. Prepare for analysis with a flow cytometer
EYRAplex assays have been validated for analysis with both Mabtech EYRA™ and flow cytometry.
- Flow cytometer requirements:
- Red laser (635–640 nm) capable of detecting at:
- 660 nm (for APC or equivalent)
- 780 nm (for APC-Cy7 or equivalent)
- Blue (488 nm), Green (532 nm), or Yellow-Green (561 nm) laser capable of detecting at:
- 575 nm (for PE or equivalent)
- Red laser (635–640 nm) capable of detecting at:
- Add 100 µl Assay buffer or PBS to each well and acquire according to your flow cytometer's flow rate recommendations.
Comparison of EYRAplex standard curves generated with data acquired with a flow cytometer and Mabtech EYRA™.