Multiplex immunoassays for flow cytometry

Multiplex immunoassays for flow cytometry

AimPlex™ 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 AimPlex 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 a 488nm laser.  The maximum emission of the bead classification dye is at 700 nm.

Similar to the principle of a sandwich ELISA, each bead population conjugated with a specific capture antibody to trap 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 min antigen and capture antibody conjugated bead incubation step, a 30 min biotinylated detection incubation step and a 20 min streptavidin-PE incubation step.

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Assay Protocol Overview

Available AimPlex Immunoassay Kit Formats

SinglePlex Kits

All available AimPlex™ immunoassays are grouped as 12-24 analytes per group.  Each analyte in a group has a unique bead region and can be multiplexed together in any combination.  Analytes in different groups may be multiplexed together if there is no conflicted on the bead regions but may have some cross-reactivity because we did not validate cross-reactivity of all the assays across different groups.

Each Single Plex Kit consists of 1 or more user selectable Analyte Kits, a Basic Kit and a sample matrix specific Diluent Kit.  Each Analyte Kit contains analyte specific Ab conjugated beads, detection antibody(ies) and corresponding antigen standard(s).  The Basic Kit has species-specific detection antibody diluent, reading buffer, wash buffer, Streptavidin-PE (SAPE), filter plate and plate seals.  The Diluent Kit contains sample-type specific Standard Diluent and sample Assay Buffer.

Premixed Multiplex Kits

Each Premixed Multiplex Kit has a pre-defined multiplex panel with premixed antibody-conjugated beads, antigens and detection antibodies.  Except the Reading Buffer (10x) and Wash Buffer (10x), all the reagents are supplied as ready-to-use.

Custom panels and assay development and custom bulk packaging

We offer custom premixed panels and custom assay development for analytes not currently available.  We will work with you to design the panels and assays.  Please contact us for pricing, lead time and details of those offerings.

We also offer custom bulk packaging, usually in 5 plate size.  Please contact us for details.

Assay Specifications
  • Sample types: Cell culture supernatant, serum, plasma, bodily fluid and tissue/cell lysate
  • Sensitivity (LOD): Majority of the assays < 10 pg/mL
  • Quantitation range: LLOQ: Majority of the assays < 20 pg/mL, ULOQ: Majority of the assays > 5,000 pg/mL
  • Standard dose recovery: 70-130%
  • Precision: Intra-assay CV: < 10%, Inter-assay CV: < 20%
  • Cross-reactivity of analytes in a panel or group: Negligible
  • Sample volume: 15 µL
Representative Data

Representative Standard Curves

Representative Mouse Cytokine Standards



Frequently asked questions

User Manual : AimPlex Multiplex Immunoassay – non-magnetic beads


Demo Video