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Multiplex ELISA Protocol by Flow Cytometry

Mutliplex ELISA Description and 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 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 ELISA Video Protocol

Multiplex ELISA Protocol Overview

Figure 1. Protocol Overview
1.) Add you Genieplex antibody bead conjugates to your well. 2.) Add your samples and standards to the wells. Incubate your samples at room temperature for 60 mins. Followed by washing the plate X3 using a filter plate vacuum system. 3) Add your biotinylated antibodies and incubate for 30 mins. Followed by washing the plate X3 using a filter plate vacuum system. 4) Add your streptavidin-PE solution and incubate for 20 mins. Followed by washing the plate X3 using a filter plate vacuum system. 5) Add Reading Buffer and read your samples on a flow cytometer. 7) Analyze your data.

Protocol Steps

Number
  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. Gently tap the plate bottom onto several layers of paper towels to remove residual buffer after the “flow-through” removal of the buffer.

  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.

  8. Add 45 µL of standards to each standard well.

9. Cover the plate with a plate seal.

  1. 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.

  2. Remove the plate seal.

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

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

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

  6. Add 25 µL of biotinylated antibody working solution to each well.

  7. Cover the plate with a plate seal.

  8. 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.

  9. Remove the plate seal.

19. Remove solutions in the wells by using the Filter Plate Washer.

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

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

  3. Add 25 µL of streptavidin-PE working solution to each well.

  4. Cover the plate with a plate seal.

  5. 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.

  6. Remove the plate seal.

  7. Remove solutions in the wells by using the Filter Plate Washer.

  8. Wash the wells twice with 100 µL 1x Wash Buffer.

  9. Gently tap the plate bottom onto several layers of paper towels to remove residual solution. 2 Add 150 µL to 300 µL of 1x Reading Buffer to each well depending on the sample loading mechanism of a flow cytometer to re-suspend the beads.

  10. Cover the plate with a plate seal.

  11. 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.

  12. Remove the plate seal.

  13. Read on a flow cytometer.