We spotted antigens at a concentration of 0.2mg/ml in phosphate buffered saline (PBS) using a BioRad VersArray Compact Microarrayer (BioRad) onto ArrayIt SuperEpoxy or SuperEpoxy 2 slides (TeleChem International, Sunnyvale, CA). we vacuum packed slides for at least 30 minutes prior to probing.
We purchased human antibody to Ro/SSA antigen (anti-Ro/SSA), human antibodies to La/SSB antigen (anti-La/SSB), mouse anti-MPO, anti-PR3, and anti-La monoclonal antibodies, and anti-Ribo-P antisera (Immunovision, Springdale, AR). W.J. van Venrooij, University of Nijmegen, the Netherlands, kindly provided the 9A9 monoclonal antibody (anti-U1A/U2B”). Antigens used in all experiments are in Supplementary Table 3. Unconjugated Affini-Pure monovalent Fab fragment goat anti-mouse IgG +IgM (H+L) and goat anti-human IgG (H+L) were from Jackson ImmunoResearch (West Grove, PA) and we used Alexa Fluor 555 or 647 carboxylic acid, succinimidyl esters (Molecular Probes, Eugene, OR according to the manufacturer’s protocol for conjugation. Cy5 and Cy3 conjugated Affini-Pure monovalent Fab fragment goat anti-mouse IgG + IgM (H+L) and goat anti-human IgG (H+L) were from Jackson ImmunoResearch (West Grove, PA), or were conjugated by Cy3 or Cy5 NHS ester (GE Healthcare, Piscataway, NJ).
Mice and treatment
We obtained BALB/c Ka mice from the breeding facility of the Department of Laboratory Animal Medicine at the Stanford University School of Medicine (Stanford, CA). At 8-10 weeks of age, female mice received a single intraperitoneal injection of either 0.5 ml pristane (Sigma-Aldrich, St. Louis, MO) or 0.5 ml sterile PBS as a control. We collected sera before injection and at 4-week intervals throughout the duration of the experiment. We obtained approval for animal experiments from the Institutional Animal Care and Use Committee.
We incubated EL4 cells at a density of 2 x 106 cells/ml in labeling medium containing the following: 45% RPMI 1640 (GIBCO, Grand Island, NY), 45% Dulbecco’s Modified Eagle Medium (DMEM) lacking L-methionine and L-cysteine (GIBCO, Grand Island, NY), 2mM glutamine (GIBCO, Grand Island, NY), 1 mM Sodium Pyruvate (GIBCO, Grand Island, NY) 5% HI-FCS, and 5% HI-FCS that had been dialyzed to equilibrium against a buffer containing 10 mM Hepes and 140 mM NaCl. We added EasyTagTM EXPRES35S Protein Labeling Mix (Perkin Elmer, Wellesley, MA) at a concentration of 0.1 mCi/ml. Cells were incubated at 37oC for 16 hours.
We lysed radiolabeled EL4 cells in 1 ml Nonidet P (NP)-40 (Sigma Chemical Co., St. Louis, MO) lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris, pH 8.0, and 1 mM EDTA). We supplemented NP-40 lysis buffer immediately before use with a 100X protease inhibitor cocktail prepared by dissolving 10 mg chymostatin, 1.5 mg leupeptin, 7 mg pepstatin A, 850 mg phenylmethylsulfonyl fluoride, 500 mg benzamidine, and 5 mg aprotonin in 50 ml of ethanol by stirring overnight. We purchased all chemicals from Sigma. We incubated cell lysates on ice for 30 min and centrifuged in a refrigerated microfuge at 13,000 rpm for 15 min. We collected and used the supernatant immediately.
Immunoprecipitation and western blot analysis
We pre-cleared lysates with 20 l of a 50% solution of protein A-Sepharose (Pharmacia, Uppsala, Sweden) in PBS and 5 g rabbit anti-mouse (RAM) IgG (Jackson ImmunoResearch, West Grove, PA) for one hour, followed by one pre-clear overnight. For precipitation experiments we used 5 micrograms of 9A9 monoclonal anti-U1A/U2B antibody, 2 l of mouse serum, or 2 l of human anti-Ribo P sera (Immunovision, Springdale, AR) along with 5 g of RAM IgG. We performed immunoprecipitations in NP-40 lysis buffer in a total volume of 500 l and rotated in a 4oC cold room for 4 hours. We harvested immunoprecipitates by centrifuging for 15 s at 13,000 rpm in a refrigerated microfuge, washed 3 times with NP-40 lysis buffer, resuspended in 2X SDS loading buffer, and boiled for 5 minutes. We fractionated the precipitates by 12% SDS-PAGE, transferred them to a nitrocellulose membrane, and exposed them for autoradiography. We blocked membranes with 5% Blotto (Bio-Rad, Inc., Hercules, CA) in PBST overnight at 4oC. We then probed the membranes with anti-Ribo P sera (Immunovision, Springdale, AR) at a dilution of 1:250 for one hour, followed by donkey anti-human (DAH) IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:5000, and developed using chemiluminescence performed according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ). For Immunoblot analysis of anti-Ribo P reactivity in mouse sera, we fractionated 100 g of purified human Ribosomal P0 antigen (Diarect, Freidburg, Germany), by 12% SDS-PAGE. We transferred the protein to nitrocellulose and blocked the membrane with 5% Blotto in PBST overnight at 4oC. We probed the membrane with individual mouse sera at a dilution of 1:250, or with anti-Ribo P human sera (Immunovision, Springdale, AR) at a dilution of 1:250, using a slot blot device (Immunetics, Cambridge, MA) for one hour, followed by incubation with HRP conjugated species-specific antibody. We visualized bands using chemiluminescence as described above.
For anti-Ribo P, anti-MPO, and anti-PR3 enzyme-linked immunosorbant assays (ELISAs), we coated 96-well Nunc Maxisorp plates (Nalgene Nunc, Milwaukee, WI) with recombinant Ribosomal P0, MPO, and PR3 antigens (Supplementary Table 3) at a concentration of 1 g/ml. We incubated wells with anti-Ribo P human sera (Immunovision, Springdale, AR) or mouse sera diluted 1:250 in 3% FCS PBST, followed by incubation with HRP-conjugated species specific secondary antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:5000. For the anti-MPO and anti-PR3 ELISAs, we used mouse sera at a dilution of 1:500 in 3% FCS PBST. We then added tetramethylbenzidine substrate (Pierce, Rockford, IL), and determined optical density values at 450 nm. We subtracted absorbances from blank wells (no serum added).