Multiplex ELISA Protocol
Multiplex ELISA Protocol
A bead-based multiplex ELISA measures up to 24 analytes in a single well by flow cytometry. This page walks through the GeniePlex workflow — three short incubations and one read, in about four hours, from as little as 15 µL of sample.
A sandwich immunoassay on a fluorescence-coded bead
A conventional ELISA measures one analyte per well. A multiplex ELISA replaces the coated plate with populations of beads that are distinguished by size and fluorescence intensity, each conjugated to a different capture antibody. Because the cytometer can tell the bead populations apart, every analyte is read separately from the same well.
Capture antibody on the bead
Each bead population carries one capture antibody, so its identity encodes which analyte it is measuring.
Biotinylated detection antibody
A second, analyte-specific antibody binds the captured target, exactly as in a sandwich ELISA.
Streptavidin-PE reporter
PE fluorescence on each bead population is proportional to the amount of that analyte in the sample.
Three short incubations, one read
The complete GeniePlex protocol runs in about 4 hours.
Capture (60 min)
Incubate sample with capture-antibody-conjugated beads, then wash.
Detect (30 min)
Add biotinylated detection antibody, then wash.
Read (20 min)
Add Streptavidin-PE, wash, resuspend and acquire on a flow cytometer.
Run your standards on the same plate as your samples. Exact volumes, incubation temperatures, standard-curve preparation and dilution series differ by panel and are specified in the technical manual and in the datasheet supplied with your kit.
Samples, instrument and expected performance
Sample volume
As little as 15 µL of sample per well.
Plex level
Quantify up to 24 targets simultaneously in one well.
Instrument
No special instrument — runs on any 488 nm (or dual 488/640 nm) flow cytometer.
Sensitivity
Below 2 pg/mL for many analytes.
Cross-reactivity
Every panel is tested for cross-reactivity in-house before release.
Sample types
Cell-culture supernatant, serum, plasma, saliva, cell and tissue lysates, BALF, pleural and peritoneal fluids and more.
Set the cytometer up before you run samples
Most failed multiplex runs are gating and compensation problems rather than assay problems. Set your instrument up against the bead populations in your panel before you commit precious samples, and keep the acquisition settings identical across the whole experiment.
Everything else you may need
Ready to run a multiplex panel?
Premixed panels ship for human, mouse, rat, canine and non-human primate samples, and custom panels are built in 7–10 days.