Microarray-based Cytokine Detection Assay

Each standard curve consisted of 12 points spanning the full range of the assay, including an assay blank of 0.1% casein in PBS. Toxins were diluted into the appropriate sample diluent. Twenty microliters of diluted toxin was applied to each well of the microarray and incubated for up to 16 hours on a gently rotating orbital shaker. Three washes were performed after each incubation step with PBS containing 0.05% Tween 20 (PBS-T). The slides were then incubated with the appropriate mix of biotinylated detection antibodies and incubated for 2 hours. The signal was enhanced using the biotinyltyramide amplification system.^{54 (link),55 (link)} The slides were incubated with 1 μg mL⁻¹ HRP-conjugated streptavidin for 30 minutes, followed by incubation for 10 minutes with 1 μg mL⁻¹ biotinyltyramide. Finally, the slides were incubated with 1 μg mL⁻¹ Cy3-conjugated streptavidin for 30–60 minutes in the dark with gentle rocking followed by a final wash and then rinsed with distilled water and dried. Experiments in the shortened assay series were incubated as indicated in the text. Cy3 fluorescence was detected by scanning slides on an LS Reloaded (Tecan, Switzerland) microarray scanner (laser: 532 nm; filter: 575 nm).