Protocol Overview

Antigen-specific binding assays are widely used in Antibody Discovery (AbD) workflows to identify antibody-secreting cells (ASC) that produce antibodies capable of recognizing a target antigen. Early identification of antigen-binding clones allows researchers to prioritize target-specific candidates, improving the efficiency of downstream characterization and reducing the time and resources required to identify high-quality antibody leads.

This protocol describes how to design, optimize, and perform an antigen-specific cell binding assay using Cyto‑Mine® Chroma, in which the antigen is expressed on the surface of a target cell.

A simplified schematic of the assay principle is shown in Figure 1. Here both an ASC as well as target cells expressing the antigen of interest, are present in the same picodroplet. As antibodies are secreted, those that recognize the antigen bind to the surface of the target cell within the droplet (Figure 1). Detection is performed using a fluorescently labelled detection antibody that binds to the secreted antibody. In the absence of antigen-specific antibody, the detection antibody will remain unbound to a cell, and the associated fluorescent signal is diffusely distributed throughout the droplet (Figure 1A)

Figure 1. Schematic representation of antigen-specific cell binding assay. A) Antibody-secreting cells are encapsulated together with target cells expressing antigen on the surface, and fluorescently-labeled detection antibody. B) Upon secretion, the primary antibody is recognized by the detection antibody and binds to the antigen on the surface of the cell. C) Binding of the antibody complex on the surface of the target cell results in translocation of some of the fluorescent detection antibody from homogenously dispersed, to localized on the cell surface, which is exhibited as an increase in peak green fluorescence signal. The fluorescence peak data (FL. Peak) is shown for three sequential droplets passing the detection point. The dotted line shows the background peak signal, with peaks appearing above this line indicating binding event.

Following secretion of the antigen-specific antibody in the droplet, the detection antibody binds to the secreted antibody (Figure 1B) and the resulting complex accumulates on the surface of the target cell, generating a localized fluorescence signal (Figure 1C).  In Cyto-Mine® Chroma, this signal can be distinguished from the diffuse fluorescence generated by cell – unbound detection antibody distributed throughout the droplet (Figure 1, FL Peak).

This protocol builds on the principles outlined above and takes advantage of the multiplexing capabilities of Cyto-Mine® Chroma to enable highly sensitive detection of antigen-specific ASC (Figure 2). In addition to the use of the fluorescent detection antibody described in Figure 1, both ASC as well as target cells are fluorescently labelled to improve signal discrimination and overall assay performance.

We also provide details of how you can adapt the protocol to exclude non-viable cells, by incorporating our Viability Probe or alternative cell-labeling strategies as appropriate for the experimental system.

Alternative protocols for detecting antibody binding to soluble antigen (FRET assay) or antigen-coated beads are available in our Knowledge Hub.

Figure 2. Schematic representation of the antigen-specific assay used in the protocol: Three types of cells were stained and prepared for co-encapsulation in picodroplets: target A431 cells (over-expressing Epidermal growth factor receptor (EGFR)) were stained with eBioscience™ Cell Proliferation Dye, eFluor™670; hybridoma cells, secreting anti-human EGFR antibodies, were stained with eBioscience™ Cell Proliferation Dye eFluor™ 450,  and an irrelevant hybridoma cell-line (secreting irrelevant anti-human TNF-α antibodies) was stained with Cell Tracker Orange CMRA. For detection of antigen-specific binding of anti-EGFR Ab to the surface of target cells, anti-mouse AF488-conjugated antibody was added to the reaction.

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