Membrane protein CD80 was used as the target, and biotin-labelled anti-CD80 antibody was used as the probe molecule (both were provided by Sysmex Corporation)39 (link). The stock solution of CD80 dispersion was 250 μg/mL and diluted to the target concentration for the experiment using phosphate buffer as the solvent. The used phosphate buffer was self-made by mixing disodium hydrogen phosphate (197-02865, Wako Pure Chemical Industries) and sodium dihydrogen phosphate (197-09705, Wako Pure Chemical Industries) at 10 mM, pH = 7.0.
Then, the anti-CD80 antibody-modified streptavidin-labelled polystyrene latex beads (2 μm in diameter, 24160, Polysciences) were used as probe microparticles. The used phosphate buffer was the same as that used for the CD80 dilution. First, streptavidin-labelled polystyrene particles were diluted to 1.148 × 109 particles/mL with 10 mM phosphate buffer. Biotin-labelled anti-CD80 antibody (undiluted concentration 1 mg/mL) was diluted to 20 μg/mL in 10 mM phosphate buffer. Equal amounts of the prepared Latex bead solution and anti-CD80 solution were mixed, and the mixture was placed in an incubator at 41.7 °C for 1 h. The beads were removed from the incubator and washed five times by centrifugation (10,000×g, 5 min) with 10 mM phosphate buffer. The prepared anti-CD80 antibody-modified beads were stored refrigerated at 4 °C.
Then, the anti-CD80 antibody-modified streptavidin-labelled polystyrene latex beads (2 μm in diameter, 24160, Polysciences) were used as probe microparticles. The used phosphate buffer was the same as that used for the CD80 dilution. First, streptavidin-labelled polystyrene particles were diluted to 1.148 × 109 particles/mL with 10 mM phosphate buffer. Biotin-labelled anti-CD80 antibody (undiluted concentration 1 mg/mL) was diluted to 20 μg/mL in 10 mM phosphate buffer. Equal amounts of the prepared Latex bead solution and anti-CD80 solution were mixed, and the mixture was placed in an incubator at 41.7 °C for 1 h. The beads were removed from the incubator and washed five times by centrifugation (10,000×g, 5 min) with 10 mM phosphate buffer. The prepared anti-CD80 antibody-modified beads were stored refrigerated at 4 °C.
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