Briefly, 50 μl of each sample or standard (0 to 5,000 pg/ml) were added into 50 μl mixtures (capture antibody-bead reagent and detector antibody-phycoerythrin reagent at a volume of 1 1) and incubated at room temperature for 3 hours away from light.
After washing the beads, protein-specific biotinylated detector antibodies were added and incubated with the beads for 1 hour.
After washing the beads, protein-specific biotinylated detector antibodies are added and incubated with the beads for 1 hr.
Culture supernatants were incubated with TNFα antibody bead concentrate, followed by biotinylated detector and streptavidin-RPE using the Biosource extracellular protein buffer kit (London, UK).
After washing, the beads were reacted with biotinylated detector antibodies and then, after additional washing, Streptavidin-R-Phycoerthrin E. After a final washing to remove all unbound material, the beads were analyzed in a Bioplex reader (BioRad, Hercules, CA, USA).
Sphero 8-peak Rainbow calibration beads (Spherotech) were used to set detector voltages each day, standardize fluorescence measurements performed on different days, and establish instrument resolution.
During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector.
To visualise the contact between the resin beads and the IRE, a focal plane array (FPA) detector collected ATR-FTIR spectroscopic images of the resin beads under load.
Movements of beads beyond the linear range of the detector (±200 nm, ±80 pN) were scored as escapes.
The beads were then incubated with a biotinylated detector antibody for one hour before incubation with streptavidin-phycoerythrin for 30 minutes.
