Arrays were blocked with 3% BSA overnight, and diluted samples was incubated on the array for 4 hours at 37 C and gentle agitation (100 RPM). Slides were then washed with PBS contain 0.05% Tween (PBST). After washing, bound antibodies were detected by Alexa 647-labeled goat anti-human IgG (Jackson Immunoresearch). Slides were then washed with PBST, spun dry in a centrifuge, and scanned.
Scan protocol
The GenePix 4000 Microarray scanner system (Molecular Devices, CA) was used for scanning.
Description
IgG D01
Data processing
We analyzed images using GenePix Pro 7.3.0.0 to measure median pixel intensity (MPI) and neighboring background pixel intensity (BPI) of individual spots. The median fluorescence intensity (MFI), representing AM-epitope specific reactivity, was calculated using the MPI minus the BPI and averaged from the triplicate spots. A minimun value was set at 100.
