Apc conjugated polyclonal goat anti human igg f ab 2 antibody

To determine mAb binding capacity to biofilm, wells containing 24 hr biofilm were blocked for 30 min with 4% BSA in PBS. After washing with PBS, wells were incubated with a concentration range of IgG1-mAbs, or Fab fragments when indicated, in PBS-BSA (1%) for 1 hr at 4°C, statically. After washing two times with PBS, samples were further statically incubated for 1 hr at 4°C with APC-conjugated polyclonal goat-anti-human IgG F(ab′)₂ antibody (Jackson ImmunoResearch, 1:500). Fab fragments were detected with Alexa Fluor 647-conjugated goat-anti-human-kappa F(ab′)₂ antibody (Southern Biotech, 1:500). After washing, fluorescence per well was measured using a CLARIOstar plate reader (BMG LABTECH).

To determine mAb binding capacity, planktonic bacterial cultures were suspended and washed in PBS containing 0.1% BSA (Serva) and mixed with a concentration range of IgG1-mAbs in a round-bottom 96-well plate in PBS-BSA. Each well contained 2.5 × 10⁶ bacteria in a total volume of 55 µL. Samples were incubated for 30 min at 4°C, shaking (~700 rpm), and washed once with PBS-BSA. Samples were further incubated for another 30 min at 4°C, shaking (~700 rpm), with APC-conjugated polyclonal goat-anti-human IgG F(ab′)₂ antibody (Jackson ImmunoResearch, 1:500). After washing, samples were fixed for 30 min with cold 1% paraformaldehyde. APC fluorescence per bacterium was measured on a flow cytometer (FACSVerse, BD). Control bacteria were used to set proper FSC and SSC gate definitions to exclude debris and aggregated bacteria. Data were analyzed with FlowJo (version 10).

Synthetic WTA (a kind gift of Jeroen Codee, Leiden University) was immobilized on magnetic beads as in van Dalen et al., 2019 (link). Shortly, biotinylated RboP hexamers were enzymatically glycosylated by recombinant TarM, TarS, or TarP with UDP-GlcNAc (Merck) as substrate. After 2 hr incubation at room temperature, 5 × 10⁷ pre-washed Dynabeads M280 Streptavidin (Thermo Fisher) were added and incubated for 15 min at room temperature. The coated beads were washed three times in PBS using a plate magnet, resuspended in PBS 0.1% BSA, and stored at 4°C. To determine CR5132 binding capacity, beads were suspended and washed in PBS/0.05% Tween/0.1% BSA and mixed with a concentration range of CR5132-IgG1 or control IgG1 in a round-bottom 96-well plate in PBS/Tween/BSA. Each well contained 10⁵ beads. Samples were incubated for 30 min at 4°C, shaking (~700 rpm), and washed once with PBS/Tween/BSA. Samples were further incubated for another 30 min at 4°C, shaking (~700 rpm), with APC-conjugated polyclonal goat-anti-human IgG F(ab′)₂ antibody (Jackson ImmunoResearch, 1:500). After washing, APC fluorescence per bead was measured on a flow cytometer (FACSVerse, BD).