Protocol: Antigen Specific anti-mouse IgG (Fc) Bead-Based Assay For Use With Cyto-Mine® Chroma
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Protocol Overview
Designed for use with our Cyto‑Mine® Chroma platform, this protocol supports confident identification of rare, high-value antibody candidates while streamlining antibody discovery workflows.
By following this protocol developed by our in-house experts, you can expect to achieve high-throughput, efficient detection and isolation of rare antigen-specific antibody-secreting cells (ASCs) within heterogeneous populations, including rare populations present at low frequency (~1%).
Learn how to create an Fc bead-based assay together with approaches designed to reduce false positive signal and background noise. This assay uses anti-mouse IgG (Fc)-coated beads to capture secreted antibodies, followed by detection using fluorescently labelled antigen.
We’ve also combined the use of a viability probe with the bead-based detection so that apoptotic or compromised cells can be excluded. This is particularly important in bead-based assays, where non-specific binding from non-viable cells can significantly increase background signal and false positives.
We used well characterized control experiments, including secretion detection and antigen-specific binding using FRET assays to verify the sensitivity and accuracy of the technique.
Direct comparison shows that the Fc bead-based assay delivers performance comparable to antigen-specific FRET, with similar sensitivity and specificity for identifying rare secreting cells.
Schematic diagram for Antigen-Specific anti-mouse (Fc) Bead-Based Assay: Antibody-secreting cells (ASC) are co-encapsulated with anti-mouse IgG (Fc)-coated beads and fluorescently labelled antigen (e.g. TNF-α). Upon secretion antibodies bind to the Fc-coated bead surface and the captured antibody subsequently interacts with the fluorescent antigen. This results in the formation of a localized fluorescent complex on the bead surface within the picodroplet.
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If yes, simply click the button, and fill in the form to download the complete protocol and start implementing antigen-specific bead-based screening on Cyto-Mine® Chroma.
Otherwise, keep reading if you need a little more information about the workflow and some of the key benefits first!
How it Works:
Step 1
Prepare viability probe and bead-based assay
Step 2
Single‑Cell Encapsulation:
- ASCs are encapsulated into uniform picodroplets with Fc beads and fluorescent antigen
Step 3
Incubation & Signal Generation:
- Secreted antibodies are captured on Fc-coated beads and bind fluorescent antigen, generating a measurable localized emission signal
Step 4
Gating & Sorting:
- Gate 1: Exclude non‑viable cells
- Gate 2: Include IgG‑secreting viable cells
Step 5
Dispense antigen-specific ASC for downstream analysis (e.g. RT-PCR)
Key Benefits:
1) Early Identification of Rare Clones
Capture low-frequency antigen-specific ASCs early in the discovery pipeline
2) High Throughput detection of Antigen-specific clones
Screen millions of cells in a single run, reducing development timelines and costs
3) Reduced false positive / background noise
Step-by-step instructions for sample preparation to reduce false positive responses during detection and isolation of clones using Cyto-Mine® Chroma
4) High Specificity
Clear, localized fluorescence signals support accurate identification of antigen-specific events
5) Flexible & Adaptable
Develop and optimize multiple assay formats on Cyto-Mine® Chroma to suit your application
Ready for the full protocol now?
Simply click the button, and fill in the form to download the complete protocol and start implementing antigen-specific bead-based screening on Cyto-Mine® Chroma.
The Next Generation: Cyto-Mine® Chroma
Automate. Accelerate. Analyze millions of cells in a single day.
Think Cyto-Mine®, but supercharged, enabling multiplexing and greater assay flexibility to fit your needs. It means that you can examine vastly greater numbers of cells — and isolate the most valuable ones— with unparalleled precision.
