Mice were immunized subcutaneously with 0.2 mg human fibrinogen (which contains citrulline modifications) (Sigma, St. Louis, MO or Calbiochem, Gibbstown, NJ) in PBS (without calcium or magnesium) emulsified with an equal volume of complete Freund’s adjuvant (CFA) consisting of incomplete Freund’s adjuvant (IFA, Sigma, St. Louis, MO) and 0.5 mg heat-inactivated Mycobacterium tuberculosis (strain H37 RA; Difco Laboratories, Detroit, MI). Twenty-one days later, the mice were boosted subcutaneously with a second injection of human fibrinogen in IFA.
Growth protocol
Female SJL/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine) and were between 7 and 9 weeks of age when experiments were initiated.
Extracted molecule
protein
Extraction protocol
At the end of the injection courses, the mice were bled for their plasma.
Label
Cy3
Label protocol
After washing with phosphate-buffered saline (PBS)/3% fetal bovine serum/0.1% Tween 20, reactive antibodies were detected using Cy3-conjugated goat-anti-human or goat-anti-mouse IgG/IgM secondary antibody (diluted 1:4,000 in PBS/3% fetal bovine serum; Jackson Immunoresearch). After incubating for 45 minutes with the secondary antibody, the slides were washed with washing solution (PBS/3 % fetal bovine serum/0.1% Tween 20).
Hybridization protocol
Synovial antigen arrays were produced using a robotic microarrayer to print peptides and proteins on ArrayIt SuperEpoxy microscope slides (TeleChem International, Sunnyvale, CA). On each slide, antigens are printed at 0.2 mg/ml in four replicates. Slides were then blocked overnight (phosphate-buffered saline (PBS), 3% fetal bovine serum, 0.1% Tween 20) and incubated for 1h at 4°C with individual mouse plasma diluted at 1:200 in PBS/3% fetal bovine serum. Slides were washed with PBS, water-rinsed, and spun dry.
Scan protocol
Slides were then scanned to determine pixel intensities for each antigen feature (Genepix 4000B; Molecular Devices, Sunnyvale, CA, USA). Genepix Pro 5.0 software (Axon Instruments) at 532 nm was used to detect IgG and IgM reactivity. Images were saved as tiff files.
Description
1:150 dilution Plasma derived from SJL mice immunized with fibrinogen emulsified in CFA or with CFA alone.
Data processing
GenePix Pro 5.0 software (Axon Instruments) was used to determine the net median pixel intensities for each antigen feature (each value is background-corrected by removing the background around each spotted protein). The median intensities for each protein were centered around 300 and log-scaled. A level of 10 digital fluorescence intensity units was set as a threshold for significant reactivity. The formula for values below 10 is log(10/300,2), and for all other values it is log(median pixel intensity/300,2). Data analysis was performed using Significance Analysis for Microarrays (SAM) software (http://www-stat-class. stanford.edu /SAM/servlet/ SAMServlet) to identify antigen features with statistically significant differences in reactivities between the experimental groups. Cluster software was then used to hierarchically group the samples and antigen features on the basis of a pairwise similarity function, and TreeView software was used to display the data as a heat map (http://rana.lbl.gov/EisenSoftware.htm).
The median intensities for each protein were centered around 300 and log-scaled. A level of 10 digital fluorescence intensity units was set as a threshold for significant reactivity. The formula for values below 10 is log(10/300,2), and for all other values it is log(median pixel intensity/300,2).
