Serum sample was centrifuged for 6min at 15000g for removing particles
Label
Alexa647
Label protocol
Antigens were spotted onto nitrocellulose-covered FAST slides (GE Healthcare) using BioOdyssey Calligrapher miniarrayer (BioRad). Features were printed in triplicates of 1:5 serial dilutions then stored at 4°C in sealed bags. Dried arrays were rinsed in PBS for 15 minutes before use, then incubated with diluted serum at 37°C for 1 hour, providing suitable conditions for complement activation. For each patient 5- times diluted serum was used in two different arrays for the detection of bound C3-IgM and IgG-C4. Sera were diluted in 5% BSA, 0.05% Tween 20, Ca and Mg supplemented Veronal buffer. Serum treated slides were washed with 0.05% Tween 20 containing PBS, then incubated in the mixture of 1:5000 diluted Alexa647-conjugated F(ab')2 fragment goat anti-human C3 antibody (Cappel) and 1:5000 diluted Cy3-conjugated F(ab')2 fragment goat anti-human IgM (mu chain speific) (Jackson ImmunoResearch) or 1:2500 diluted FITC-conjugated goat anti-human C4 (Cappel) and 1:2500 diluted APC-conjugated F(ab')2 fragment goat anti-human IgG (gamma chain specific) (Jackson ImmunoResearch).Labeling with fluorescent antibodies was carried out at room temperature for 30 min in 5% BSA and 0.05% Tween 20 containing PBS. Following washing in 0.05% Tween 20 containing PBS, arrays were dried and scanned.
Hybridization protocol
Probe: Alexa647-conjugated F(ab')2 fragment goat anti-human C3 antibody
Scan protocol
Arrays were scanned on Typhoon Trio+ imager (Amersham Bioscience) (Excitation:633nm ; Emission: 670BP, PMT:420 Mode:Fluorescence, Sensitivity:Normal, FocalPlane=+3 mm). Data were analyzed with GenePix Pro 6 software (Molecular Devices, Sunnyvale, CA, USA).
Data processing
Local background of spot was substracted from the value of signal. The value of signal is set to zero if it is lower than 2*SD of local backgrounds on the given chip. For interassay comparison, data were normalized to the mean of 5 times diluted Protein G control material (ID_REF: #52, #53, #54). Normalization procedure was carried out with Microsoft Office Exel software
