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Multiplex Assays

Multiplex Assays

"It’s like doing 24 different ELISA in every well!"

GeniePlex Multiplex Assays allow researchers to quantify up to 24 analytes in as little as 15μL of sample by Flow Cytometry!

GeniePlex Multiplex Protocol

Standard curves of Human 18-Plex

GeniePlex Multiplex Features

  • Quantitative | Measure up to 24 analytes simultaneuosly

  • Less Volume | 15μL of sample per well

  • No Special Instrument Required | Run on any flow cytometer

  • High Sensitivity | < 2 pg/mL for many analytes

  • Rapid Protocol | Assay Time approx. 4 hours

  • Precise Technology | Intra-Assay CV: <10%, Inter-Assay < 20%

  • Easy Analysis | Easy analysis using commonly available software

GeniePlex Multiplex Benefits

24-plex cAb beads analyzed on a flow cytometer equipped with both a 488nm and 640nm laser

Custom GeniePlex Panels

At Assay Genie, we specialise in custom multiplex panel development.

We develop bespoke panels tailored towards researcher's experiments in as little as 7-10 business days!

All of our custom panels are fully validated & tested for cross reactivity before they leave the lab. Fill out a custom form below for a human, mouse, rat, NHP or canine custom panel!

Click Here to Create a Custom GeniePlex Panel!

Activin A Flt-3 Ligand/Flt-3L/Flk-21 LIF
Adiponectin Follistatin/FST Lipocalin-2/NGAL
AFP Galectin-1/Galaptin L-selectin/LECAM-1/sCD62L
ALCAM/sCD166 Galectin-9 Lymphotactin/XCL1/SCYC1
Amphiregulin GASP-1/WFIKKNRP M-CSF/CSF1
ANGPT-1/Angiopoietin-1 G-CSF/CSF-3 MIA/CD-RAP
Artemin/ARTN/Neublastin GDF-15/MIC-1/PTGFP Midkine/MK1/NEGF2
BCMA/TNFRSF17/sCD269 GDNF/ATF-1 MIF
BDNF GM-CSF/CSF-2 MMP-1/CLGN (Total)
Betacellulin/BTC Gp130/sCD130/IL6ST MMP-13/Collagenase 3(Total)
BIGH3/TGFBI/βIG-H3 Granzyme A/GZMA/CTLA3 MMP-2/Gelatinase A
BLAME/SLAMF8/sCD353 Granzyme B MMP-3/Stomelysin-1 (Total)
BlyS/BAFF/sCD257/TNFSF13B HGF/Hepatopoietin A MMP-7/PUMP-1 (Total)
BMP-2 HGF/Hepatopoietin A MMP-8/Collagenase 2 (Total)
BMP-4 ICAM-1/sCD54 MMP-9/GELB (Total)
CA125/MUC16 ICAM-3/sCD50 MPO
CA15-3/MUC1 IFN-gamma NCAM-1/sCD56
CA19-9 IFN-α2 NOV/CCN3/IGFBP-9
CA50 IFN-β NRG1-beta1/HRG1-beta1
CCL1/SCYA1/I-309 IgA OPG/Osteoprotegerin/TNFRSF11B
CCL11/SCYA11/Eotaxin IgE OPN/Osteopontin
CCL13/SCYA13/MCP-4 IGFBP-1 OSM/Oncostatin M
CCL14/SCYA14/HCC-1 IGFBP-2 PAPPA/PAPP-A
CCL15/SCYA15/MIP-1 delta IGFBP-3 PD-1/sCD279
CCL16/SCYA16/HCC-4 IGFBP-5 PDCD1LG1/PD-L1/sCD274
CCL17/SCYA17/TARC IGFBP-6 PDCD1LG2/PD-L2/sCD273
CCL18/SCYA18/PARC IGFBP-7 PDGF-AA
CCL19/SCYA19/MIP-3β IgG1 PDGF-AB
CCL2/SCYA2/MCP-1 IgG2 PDGF-BB
CCL20/SCYA20/MIP-3α IgG3 PECAM-1/sCD31
CCL21/SYCA21/6Ckine IgG4 Perforin/PFP/PRF1
CCL22/SCYA22/MDC IL-10/CSIF PIGF/PIG-F
CCL23/SCYA23/MPIF-1 IL-11 Pro-BDNF
CCL24/SCYA24/Eotaxin-2/MPIF-2 IL-12/IL-23p40 Procalcitonin/PCT
CCL25/SCYA/TECK IL-12p70 P-selectin/LECAM-1/sCD62P
CCL26/SCYA26/Eotaxin-3 IL-12p70 RAGE/AGER/sRAGE
CCL27/SYCA27/CTACK IL-13 Reg3A/PAP2/PAPPAP-H/PBCGF
CCL28/SYCA28/MEC IL-15 Resistin/ADSF
CCL3/SYCA3/MIP-1 alpha IL-16/LCF SAA/Serum Amyloid A
CCL4/SYCA4/MIP-1 beta IL-17A/CTLA-8 SCF/MGF/KIT-Ligand
CCL5/SCYA5/RANTES IL-17C SLAMF4/2B4/NAIL/sCD244
CCL7/SCYA7/MCP-3/MARC IL-17F SLPI/ALP/BLPI
CCL8/SCYA8/MCP-2 IL-18 ST2/IL-1R4/IL-1RL1
CD14/sCD14 IL-19 Survivin/BIRC5
CD163/M130/sCD163 IL-1R2/sCD121b TGF-alpha/TGFα/TGFA
CD73/NT5E/5'-Nucleotidase IL-1RA TGFβ1
CEA/CD66e/CEACAM5 IL-1RI/sCD121a THPO/Thrombopoietin
CEACAM-1/sCD66a IL-1α TIM-1/KIM-1/HAVCR
CNTF IL-1β TIM-3/HAVCR2
CRP/C-Reactive Protein IL-2 TIMP-1
CX3CL1/Fractalkine IL-20/IL-10D TIMP-2
CXCL1/MGSA/GROα IL-21 TL1A/TNFSF15/VEGI
CXCL10/IP-10 IL-22/IL-TIF TNF-alpha/TNFα
CXCL11/I-TAC IL-23 TNF-beta/TNFSF1/LTA
CXCL12/SDF-1 IL-23/IL-23p19 TNFRSF10C/sCD263/sTRAIL-R3
CXCL13/BCA-1 IL-24 TNFRSF13B/sCD267/TACI
CXCL14/BRAK IL-25/IL-17E TNFRSF14/sCD258/LIGHT
CXCL16/GCP-3 IL-27 TNFRSF1A/sCD120a/sTNF RI
CXCL2/GROβ/MIP-2α/GRO2 IL-27 TNFRSF1B/sCD120b/sTNF R2
CXCL4/PF4 IL-28A/IFN-lambda-2 TNFRSF5/sCD40
CXCL5/ENA-78 IL-29/IFN-lambda-1 TNFRSF6/sCD95/sFasR
CXCL6/GCP-2 IL-2RA/sCD25 TNFRSF9/sCD137/s4-1BB
CXCL7/NAP-2 IL-3 TNFSF10/sCD253/TRAIL
CXCL9/MIG IL-31 TNFSF11/sCD254/sRANKL
Cystatin C/CST3 IL-32 TNFSF13/sCD256/APRIL
E-Cadherin/CDH1/sCD324 IL-33 TNFSF14/sCD258/sLIGHT
EGF IL-34 TNFSF5/sCD154/sCD40L
EGFR/ErbB1 IL-35 TNFSF6/sCD95L/sCD178/sFasL
EG-VEGF/PROK1/PK1 IL-4 TNFSF8/sCD153/sCD30L
Elafin/PI3/Trappin2 IL-5 TSLP
Endoglin/sCD105 IL-6 TWEAK/TNFSF12/Apo3 Ligand
Endostatin IL-6Rα/sCD126 Uteroglobin/CC10/CC16
EpCAM/MK-1/TROP1/sCD326 IL-7 VAP-1/AOC3
EPO-R/Erythropoietin Receptor IL-8/CXCL8 VCAM-1/sCD106
E-selectin/ELAM-1/sCD62E IL-9 VEGF-A
Ferritin Insulin VEGF-C
FGF-1/FGF-acidic/HBGF-1 Keratin 19/KRT19/CK-19 VEGF-D/FIGF
FGF-19 KGF/FGF-7 VEGFR1/Flt-1
FGF-2/FGF-basic/HBGH-2 Klotho/KLA/KL VEGFR2/Flk-1/sCD309/KDR
FGF-4 LAP (TGF-β1)/Latent TGF-β1 β-NGF/NGFB
FGF-9/HBGF-9/GAF Leptin/LEP

GeniePlex Success Stories

See some of the institutions we have partnered with and some of our recent customer success stories!

Results from Custom Canine 10-Plex assay

GeniePlex Analysis & Results

At Assay Genie we do the data analysis for you for free!

Send us your data and we will send back a data report in less than 12 hours!

What do your results look like? Check out the results from a custom canine 10-Plex assay below!

GeniePlex Training Sessions

We provide 3 FREE Training Sessions included with every GeniePlex assay to make sure you get the best results possible!

Session Details

1.

The day before you run your samples we will join via zoom to set up an acquisition protocol on your flow cytometer. This allows you to find the bead populations and save the settings for the sample run!

2.

We know samples are precious! So the day of your sample run we'll join to run some standards and a few samples to make sure your experiment is running as smoothly as possible.

3.

Free Data Analysis! Send us the FCS files and we'll send back a full data analysis report in less than 12 hours. See an example of the report you receive above. If you want to do it yourself we can provide a 30-minute analysis workshop to show you how it's done.

Free Consultation

Have a question? Schedule a free consultation with one of our GeniePlex experts!

Book a time slot for a video consultation using the calendar.

Alternatively you can sumbit a contact form!

Multiplex vs. ELISA

Multiplex Assays require less sample volume, less plates, less time and have greater sensitivity!

Click to enlarge!

GeniePlex Multiplex Immunoassays show excellent correlation with Luminex xMAP technology

GeniePlex vs Luminex?

Our GeniePlex Multiplex Assays give excellent performance versus Luminex technology.

The Human TH1/TH2/TH17 7-Plex ELISA and a Luminex multiplex assay were used to assay cell culture supernatant samples from human PBMCs treated with various reagents.

GeniePlex Multiplex Workflow

Schematic of multiplex workflow. Click to enlarge!

GeniePlex Multiplex Assay Protocol

Protocol Steps

Step Protocol

1.

Prepare the filter plate template. Mark the standard, sample and blank wells. Standards and samples should be run in duplicates or triplicates. If the whole plate will not be used, seal the unused well with a plate seal.

IMPORTANT: Place the filter plate on top of the filter plate lid during the entire assay process to prevent touching the plate bottom on any surface.

2.

Vortex working bead suspension for 15 seconds.

3.

Add 45 µL of capture bead working suspension to each well. NOTE: Save the remaining capture bead working suspension and store at 2-8°C with light protection. It can be used for setting up acquisition parameters on the flow cytometer.

4.

Remove buffer in the wells by using the “flow-through“ Filter Plate Washer connected to a vacuum source that has been adjusted according to the Filter Plate Washer Instructions.

5.

Remove buffer in the wells by using the “flow-through“ Filter Plate Washer connected to a vacuum source that has been adjusted according to the Filter Plate Washer Instructions.

6.

Add 30 µL of CCS, SPB or TL Assay Buffer to each sample well.

NOTE: Cell culture supernatant samples can be run without diluting in Assay Buffer if very low levels (less than 20 pg/mL) of cytokines are expected. If it is the case, skip this step and add 45 µL of cell culture supernatant samples to each sample well in Step 7.

7.

Add 15 µL of samples to each sample well. Add 45 µL of standards to each standard well. Cover the plate with a plate seal.

8.

Incubate on the shaker (set at 700 rpm) for 60 min at room temperature. Protect from light by wrapping the filter plate in aluminum foil.

9.

Remove the plate seal. Remove solutions in the wells by using the Filter Plate Washer connected to a vacuum source.

10.

Remove solutions in the wells by using the Filter Plate Washer connected to a vacuum source.

11.

Wash the wells three times with 100µL 1x Wash Buffer using the Filter Plate Washer.

12.

Gently tap the plate bottom onto several layers of paper towels to remove residual buffer on the plate bottom after the last wash.

13.

Add 25µL of biotinylated antibody working solution to each well. Cover the plate with a plate seal.

14.

Incubate on the shaker (set at 700 rpm) for 30 min at room temperature. Protect from light by wrapping the filter plate in aluminum foil.

15.

Remove the plate seal. Remove solutions in the wells by using the Filter Plate Washer. Wash the wells three times with 100 µL 1x Wash Buffer using the Filter Plate Washer.

16.

Gently tap the plate bottom onto several layers of paper towels to remove residual buffer on the plate bottom after the last wash.

17.

Add 25µL of streptavidin-PE working solution to each well. Cover the plate with a plate seal.

18.

Incubate on the shaker (set at 700 rpm) for 20 min at room temperature. Protect from light by wrapping the filter plate in aluminum foil.

19.

Remove the plate seal. Remove solutions in the wells by using the Filter Plate Washer. Wash the wells twice with 100 µL 1x Wash Buffer.

20.

Gently tap the plate bottom onto several layers of paper towels to remove residual solution. Add 150µL to 300µL of 1x Reading Buffer to each well depending on the sample loading mechanism of a flow cytometer to re-suspend the beads. Cover the plate with a plate seal.

21.

Place the plate on the microtiter shaker and shake for 30 seconds at 700 rpm.

NOTE: If the flow cytometer has no 96-well plate loader and more than 200 µL of 1x Reading Buffer is needed to re-suspend the beads, do not shake the plate. Re-suspend the beads in each well by pipetting up and down 6–8 times with a P200 pipette then transfer to a test tube for acquisition. Remove the plate seal. Read on a flow cytometer.

What is a Multiplex Assay?

Several antibody-based assay platforms have been developed as alternatives to ELISA for the quantitative analysis of analytes in a single sample at the same time, also known as multiplex assays. The most common platform for multiplex immunoassays uses a flow-based technology and antibody-coated beads. The beads can be used to simultaneously measure multiple analytes in a single sample.

In the multiplex assay, antibodies specific to particular analytes are connected to a set of beads within the assay. Another antibody is then added, which has a fluorescent reporter attached. Each set of beads has an associated colour, which results in the simultaneous detection of multiple analytes in a single sample. The detection of analytes can be read by a flow cytometer as the beads are detectable by various fluorescent signatures. The amount of analytes can be quantified based of the number of different bead colours detected.

How Multiplex Works

The Assay Genie Multiplex utilizes multiple bead populations differentiated by size and different levels of fluorescence intensity. With multiple sizes of beads and multiple levels of fluorescence intensity in each bead size, the multiplex technology can measure up to 24 analytes simultaneously in a single reaction. The Multiplex ELISA requires as little as 15μL of sample.  The bead populations in the reaction are determined by a flow cytometer equipped with either a single 488nm laser or dual 488nm and 633/640nm lasers. The maximum emission of the bead classification dye is at 700 nm.

Bead-based immunoassays are similar to the principle of a sandwich ELISA, having each bead population conjugated with a specific capture antibody trapping the protein of interest, such as a cytokine, in the sample. The amount of the analyte captured is detected via a biotinylated antibody against a secondary epitope of the protein, followed by a streptavidin-R-phycoerythrin treatment. The fluorescent intensity of R-phycoerythrin on the beads is quantified on a flow cytometer.

Concentrations of a protein of interest in the samples can be obtained by comparing the fluorescent signals to those of a standard curve generated from a serial dilution of a known concentration of the analyte.

The assay procedure consists of a 60-minute antigen and capture antibody conjugated bead incubation step, a 30-minute biotinylated detection incubation step and a 20-minute streptavidinPE incubation step.

Multiplex Troubleshooting

Problem Solutions

Low level count

Possible cause Recommended actions

Beads aggregate

Vortex stock and working bead
suspensions well before pipetting.

Beads settle on the well bottom

Shake plates at 700 rpm for 30 seconds
prior to acquisition or re-suspend the
beads in a well by pipetting up and down
6–8 times with a P200 pipette prior to
transferring to a sample tube for acquisition.

Vacuum too strong

Adjust the vacuum pressure so that 100
µL of 1x Wash Buffer in the wells can be
clear in 3-5 second.

Low assay signal or sensitivity

Possible cause Recommended actions

Standard not
reconstituted well

Standard(s) should be incubated on ice
for 5min after the addition of standard
diluent.

Incubation time too
short

Follow recommended incubation time in
each step.

Excess exposure to
light

During incubation, cover the plate with
aluminum foil to minimize exposure of the
beads to light.

High Background

Possible cause Recommended actions

Well-to-well
contamination

Change pipette tips after every transfer.
Remove plate seal carefully and avoid
contents from one well to mix with another.

Low Precision

Possible cause Recommended actions

Poor pipetting
precision

Use calibrated pipettes.

Contamination
from adjacent wells

Avoid well-to-well contamination during
pipetting and removal of plate seal.

Clogged filter plate

Possible cause Recommended actions

High lipid content
in the serum,
plasma or bodily
fluid samples

Centrifuge the samples at 10,000 x g for
10 min at 2-8°C. Collect the serum,
plasma or bodily fluid fraction.


Additional Resources