Multiplex ELISA

Cytokine Bead Array for Multiplex Analyte Detection by Flow Cytometry

GeniePlex is a bead-based multiplex immunoassay technology. It enables the simultaneous and quantitative detection of up to twenty-four analytes in as little as 15μl sample. The GeniePlex immunoassay can be carried out on an existing flow cytometer - the purchase of another expensive instrument is not required.

  • Measure up to 24 analytes by Flow Cytometry
  • Only 15μL of sample required
  • Pre-mixed panels for Human, Mouse, Rat, Canine & Non-Human Primate
  • Create your own custom panel
GeniePlex Multiplex Assay

Figure 1: Schematic of GeniePlex workflow

  • Use Your Own Lab: Assays can be run on most validated flow cytometers (PE & either PE-Cy5 or APC detectors).
  • Free Software: Analyse with commonly available software such as FCAP Software v3.0. Otherwise, send us your data!
  • Customization: Can’t find your analyte in our extensive list? Ask our team of experienced scientists to help build your assay.

What is GeniePlex Multiplex ELISA?

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.

Genieplex kits allow researchers to multiplex up to 24 analytes using as little as 15μL by Flow Cytometry!

Figure 2: Standard curves of Human 18-Plex

ELISA Genie Multiplex Assay Principle

The ELISA Genie, GeniePlex multiplex assay technology 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 GeniePlex technology can measure up to 24 analytes simultaneously in a single reaction. The GeniePlex multiplex assay 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.

Figure 3: 24-plex cAb beads analyzed on a flow cytometer equipped with both a 488nm and 640nm laser

Features & Benefits

  • Analyze: Multiplex 2-24 analytes in a single sample!
  • Fast: 2 hour protocol!
  • Sensitive: Measure as low as <10 pg/ml of each analyte
  • Dynamic: Lower limit < 20 pg/mL | Upper limit > 5,000 pg/mL
  • High Precision Intra-assay CV: < 10% | Inter-assay CV: < 20%
  • Low Volumes: Use as little as 15μl of sample
  • Validated: All assays fully tested for cross-reactivity in our lab

GeniePlex or Luminex?

GeniePlex gives excellent performance versus Luminex technology. GeniePlex Human TH1/TH2/TH17 7-Plex assay and a Luminex multiplex assay were used to assay cell culture supernatant samples from human PBMCs treated with various reagents.

GeniePlex versus Luminex data

Figure 4: GeniePlex Multiplex Immunoassays show excellent correlation with Luminex xMAP technology

GeniePlex vs ELISA

GeniePlex ELISA

Sample Volume

15µL

50-700µL

96-well plates

1 plate

7 plates

Time

~4 hrs

~ 4-7 hrs

Assay Range

2-5000pg/mL

2-1500pg/mL

Figure 5: Number of analytes available with GeniePlex by species

Over 400 Analytes to measure!

  • Comprehensive Choice of Targets
  • Up to 400 assays & custom formats available for human, mouse, rat, porcine, canine and primates
  • Wide Range of Sample Types
  • Cell culture supernatants, saliva, plasma, cell/tissue lysates, serum, BALF, pleural and peritoneal fluids & more
  • Multiple Formats: 32-well and 96-well sizes available
  • Key Premixed Panels
  • We have designed key panels to measure Cytokines, Chemokines, Inflammation, Th1/Th2 & Th1/Th2/Th17

Custom Panels Available!

Create your own custom multiplex assay with over 400 analytes to choose from!

At ELISA Genie we can develop custom assays to meet your research needs within 20 days! To find out more information about our developing your own custom panel, please click here to get in touch!

Multiplex ELISA 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.


Multiplex ELISA 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.


Multiplex ELISA Resources