AimPlex Flow Cytometry Based Multiplex Cytokine Assays

How It Works

Insight Biotechnology is extremely pleased to distribute AimPlexTM multiplex flow cytometry based cytokine assays from innovative US based company YSL Bio. Feel free to contact our PhD level technical team to discuss this highly customisable technology and we will tailor a quote towards the needs of your project. Telephone +44(0) 208 385 0303 or email info@insightbio.com.

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 traps 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.

AimPlex Bead Based Immunoassays Diagram

Assay Protocol Overview

Assay Protocol Overview Diagram

Available AimPlex Immunoassay Kit Formats

Single Plex 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 but may have some cross-reactivity because cross-reactivity of all the assays across different groups was not validated.  We offer custom multiplex assay panel design and development.  Please contact us for details including pricing and lead time.

Note: Bead set assignment may vary in a panel. Refer to packaging/product inserts and kit labels for details.

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.  Some of the single-plex kits are available in both 96- and 32-test sizes.

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.  Most of the premixed multiplex kits are available in both 96- and 32-test sizes.

Note: Bead set assignment may vary in a panel. Refer to packaging/product inserts and kit labels for details.

Custom panel 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 Standard Curves Human DiagramRepresentative Standard Curves Mouse Diagram

Analytes LOD (pg/mL) LLOQ (pg/mL)
Human IFNg <3 <10
Human IL-2 <3 <10
Human IL-4 <1 <3
Human IL-6 <5 <10
Human IL-10 <2 <5
Human IL-17A <3 <10
Human TNFa <1 <2
Analytes LOD (pg/mL) LLOQ (pg/mL)
Mouse IFNg <0.5 <2
Mouse IL-2 <2 <5
Mouse IL-4 <0.5 <2
Mouse IL-6 <3 <10
Mouse IL-10 <2 <5
Mouse IL-17A <1.5 <5
Mouse TNFa <0.5 <2

Representative Mouse Cytokine Standards


Representative Mouse Cytokine Standards 5,000pg/mL DiagramRepresentative Mouse Cytokine Standards 1,667pg/mL DiagramRepresentative Mouse Cytokine Standards 556pg/mL Diagram

Representative Mouse Cytokine Standards 185pg/mL DiagramRepresentative Mouse Cytokine Standards 62pg/mL DiagramRepresentative Mouse Cytokine Standards 21pg/mL Diagram

Representative Mouse Cytokine Standards 7pg/mL DiagramRepresentative Mouse Cytokine Standards 0pg/mL Diagram

Human PBMC

Cell culture supernatant samples from PBMCs treated and untreated (control) with PMA + Ionomycine (P+I), anti-CD3 (CD3) and LPS were tested by the AimPlex Th1/Th2/Th17 7-Plex Assays.

Human PBMC Diagram

 

Correlation with Luminex* Assays

Cell culture supernatant samples from PBMCs treated with various reagents were assayed with AimPlex Th1/Th2/Th17 7-Plex assays and a commercially available Luminex multiplex assay kit.


Correlation with Luminex Assays IFNg DiagramCorrelation with Luminex Assays IL-2 DiagramCorrelation with Luminex Assays IL-4 Diagram


Correlation with Luminex Assays IL-6 DiagramCorrelation with Luminex Assays IL-10 DiagramCorrelation with Luminex Assays IL-17A Diagram


Correlation with Luminex Assays TNFa Diagram


Correlation with CBA* Assays

Human normal and abnormal serum samples were measured with both AimPlex® and CBA assays and analyzed on a BD FACSCanto II.


Correlation with CBA Assays G-CSF DiagramCorrelation with CBA Assays IL-6 DiagramCorrelation with CBA Assays IL-10 Diagram


Correlation with CBA Assays TNFalpha Diagram

 

Mouse Serum Chart

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AimPlex Assay Procedure Overview Video

The video demonstrates the steps and techniques required to properly perform the AimPlex assay.  The use of the EZPrep Filter Plate Washer is highlighted.  Some steps are not shown for brevity.

Feel free to contact our PhD level technical team to discuss this highly customisable technology and we will tailor a quote towards your specific objectives. Telephone +44(0) 208 385 0303 or email info@insightbio.com.