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.
9. Cover the plate with a plate seal.
19. Remove solutions in the wells by using the Filter Plate Washer.