Extended Data Fig. 4: Generation of anti-OVA antibodies from encapsulated mouse bone marrow plasma cells and analysis of confocal images.

(a) Analysis of confocal microscopy image of an encapsulated OVA-specific bone marrow plasma cell showing the spatial distribution of CD138 (AF647), IgG (AF405) and OVA (AF555) within the VHH-functionalized BG-agarose bead. The graphs display the pixel intensity along the red line from left to right. IgG and OVA signal intensity decrease as a function of distance from the encapsulated cell. A representative image of over 40 images obtained from two biological replicates is shown. Additional confocal images can be found in Supplementary Fig. 4. (b) Comparison of the IgG signal distribution of a hydrogel bead containing a cell with a neighboring empty bead. The cross-section for analysis is indicated by a red line. A representative image from two biological replicates is shown. Additional confocal images can be found in Supplementary Fig. 4. (c) Mouse immunization and analysis scheme. Bone marrow plasma cells (CD138⁺) were magnetically enriched. Cells were encapsulated into VHH-functionalized BG-agarose. OVA-specific plasma cells were sorted with fluorescently labeled monomeric OVA (OVA-AF647). (d) Comparison of IgG signal originating from plasma cells in BG-agarose beads incubated with (+VHH) or without VHH–SNAP–FLAG (−VHH; different experiment than shown in Fig. 1). The plots show 527 (−VHH) and 1,280 (+VHH) events at 2% contour level. (e) Representative gating strategy for identifying OVA-specific plasma cells from bone marrow. Live plasma cells inside hydrogel beads were gated as live/FLAG⁺/CD138⁺ (VHH–SNAP contains a FLAG tag). (f) Determination of binding affinity of mOVA1 and mOVA2 by biolayer interferometry: mOVA1: K_D = 2.36 ± 0.02 nM, χ² = 0.0705, R² = 0.985. mOVA2: K_D = 0.684 ± 0.004 nM, χ² = 0.0388, R² = 0.9919. Tested OVA concentrations: 1.02–16 nM.