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Julie Brouchon

Microfluidic-based technology to isolate B cells secreting functional monoclonal antibodies from human samples

We are developing a microfluidic technology enabling high-throughput identification and isolation of individual cells that secrete desired antibodies, followed by rapid identification of the paired heavy- and light-chain sequences encoding these antibodies of interest. This powerful technology can be applied to cells from any species, including human, and provides single cell resolution and high throughput, which are essential for analysis of large populations of antibody-secreting cells. A complete description of the microfluidic assay and a proof of principle are shown in Figure 1a and 1b. Our goal while developing this technology is to identify antibodies for immuno-therapy to fight HIV infection or cancer. 

Development of a microfluidic assay compatible with interrogation and sorting by Fluorescence Activated Cells Sorting (FACS)
We are now focusing on improving the accuracy and throughput of the assay (Fig 1a) by integrating Fluorescence Activated Cells Sorting (FACS) into our workflow. FACS is one of the most powerful and well-established sorting technologies, but the instruments are designed for use with aqueous-based solutions. So we are currently working on converting the water-in-oil droplets into alginate hydrogels. The hydrogel network has a pore size of ~20 nm so the capture bead, which is the read-out for antibody activity, and the cell, remain linked after breaking the emulsion as shown in Figure 1b. The hydrogel micro-particles containing cells and the beads can now be sorted using FACS. Another important capability of the hydrogel is that all the molecules that are not bind to the bead can be washed away. You can clearly see this by comparing the residual fluorescence in the droplet versus the micro-particle. That can be essential for some assay and in the presented case it significantly increases the signal (fluorescence of the bead) over noise (fluorescence of the droplet or gel) ratio. We hope to apply this method to human patient samples to isolate cells that secrete antibodies against viral proteins or cancer-related epitopes. 

Principle of antibody binding assay
Figure 1. Principle of antibody binding assay. Schematic model depicting the method. B cells (orange) are encapsulated in an aqueous based droplet surrounded by oil in a microfluidic chips. Each droplet contains beads (grey) coated with anti-human antibodies (yellow) and fluorescent–labeled detection probe P (green). The fluorescent–labeled detection probe is a protein or an oligo derived from a virus or a cancer cell, for example. This probe determines which type of antibodies the assay will identify. After incubation for 60 min at 37°C, beads that are co-encapsulated with a cell producing an antibody able to bind the fluorescent protein become highly fluorescent due to the capture of secreted antibodies (orange) on the bead by the anti-human antibody and binding of the fluorescent–labeled detection protein to the captured antibodies. Beads that are co-encapsulated with a cell producing an antibody not able to bind the fluorescent protein are not fluorescent.
Principle of antibody binding assay
Figure 2. Principle of antibody binding assay. Microscopy of encapsulated cells in aqueous droplets (left images) and in alginate micro-particles (right images). Hybridoma cells that secrete mouse antibody are unlabeled (top two images) and negative control cells are labeled with a red fluorescent live dye (bottom two images). Droplets and gels also contain fluorescent-green-labeled anti-mouse detection antibodies and beads coated with anti-mouse-Fc capture antibodies. Beads co-encapsulated with the antibody-secreting cell become highly fluorescent due to the bead-capture of secreted antibodies and binding of the detection antibodies to those captured antibodies. As expected, beads co-encapsulated with a negative control cell are not fluorescent. Because the droplet contains alginate and divalent ions, cells and beads stay co-encapsulated in alginate micro-particles after the emulsion is broken.