Patients and controls. Serum samples from 152 Japanese patients were studied: 29 from patients with active AS (New York criteria) (27 men/2 women) [mean age 42 years, range 20-69; mean erythrocyte sedimentation rate (ESR) 44.9 mm/h (standard error: SE ± 3.7), mean C-reactive protein (CRP) 24.8 mg/1 (± 3.1)]; 23 patients with inactive AS (21 men/2 women) [mean age 42 years, range 25-68; mean ESR 12.2 mm/h (± 2.1), mean CRP 3.9 mg/1 (± 1.1)]; 30 patients with active RA (American Rheumatism Association criteria) (5 men/25 women) [mean age 51 years, range 23-70; mean ESR 77.7 mm/h (± 5.9), mean CRP 40.2 mg/1 (± 5.2)]; 20 patients with probably active RA (5 men/15 women) [mean age 55 years, range 30-77; mean ESR 38.3 mm/h (±3.4), mean CRP 5.9 mg/1 (± 1.3)], and 50 healthy controls (25 men/25 women) (mean age 47 years, range 30-60), control samples were supplied by the Red Cross Blood Center, Otsu, Japan. Active patients were deemed to be those who had both ESR > 20 mm/h and serum CRP > 10 mg/1. Probable active patients were considered those with at least one of these variables elevated and inactive patients were those with both ESR < 15 mm/h and serum CRP < 10 mg/110.
All patients with AS except one inactive patient were HLA-B27 positive. The percentage positive of active RA associated tissue types, DR1 or DR4, was 93.3%, and 40% had both DR1 and DR4 alleles. Control subjects were not tissue typed.
Bacterial cultures. K. pneumoniae (K54), P. mirabilis, and Escherichia coli were urinary tract isolates obtained from the Department of Microbiology, King's College, London. Bacterial cultures were grown aerobically in 250 ml conical flasks on an orbital shaker for 16-18 h at 37 °C in nutrient broth (Oxoid: 25 g/1) to obtain a stationary phase culture. Cells were harvested by centrifugation (MSB 18, 6 x 250 ml rotor) and washed 3 times in 0.15 M phosphate buffered saline (PBS), pH 7.4. The cells were resuspended in 20 ml of PBS and then diluted in carbonate buffer, 0.05 M, pH 9.6, to give an optical density (OD) reading of 0.25 on the spectrophotometer (Corning, Model 258), which is equivalent to 6 x 10s cells/ml. Peptide synthesis. Three 16 mer peptides were constructed: the HLA-B27 sequence (residues 67-83) CKAKAOTdredLRTLL. the pullulanase-D (pulD) secretion protein sequence (residues "590-605) RPTVIR-jinlfiYRQASS, and a control peptide sequence made from a scrambled sequence of the pulD peptide, RPTVRSDIDYRQAESR. All synthetic peptides were prepared by solid phase synthesis; purity assays were performed by
high performance liquid chromatography. They had a purity of at least 90% EUSA. All serum samples were tested against 3 bacterial antigens and 3 synthetic peptides by ELISA. All assays were carried out under code, so that the status of each serum sample under investigation was not known to the tester. Briefly, bacteria (100 pi or 100 ul of 5 ug/ml peptide solution) were fixed in polystyrene microtiter ELISA plates (Dynatech, McLean, VA USA) overnight at 4"C. After absorption and washing with PBS containing 0.1% (v/v) Tween 20 (Sigma, St. Louis, MO, USA), the plates were saturated with 0.5% (w/v) bovine serum albumin (Sigma) PBS-Tween 20 and incubated for 1 h at 37°C to block nonspecific binding. Serum samples (1/200 dilution in PBS-Tween on bacterial studies and 1/50 dilution in PBS-Tween on peptide studies) were added to the plates, incubated for 2 h at 37°C, followed by washing with PBS-Tween and peroxidase conjugated rabbit antihuman class specific IgG, IgA. or IgM (DAKO Ltd.) diluted 1/500 in PBS-Tween was added and the plates incubated for 2 h at 37°C. After washing, the substrate solution, 0.5 ml 2.2'-azinobis (3-ethyIbenz-thi-azoline-6-sulphonic acid) ABTS (Sigma) in citrate/phosphate buffer, pH 4.1, containing 0.98 mM H2O2 (Sigma). was added to each well. Development of the plates took place at room temperature in the dark for 20 min. The reaction was stopped with 2 mg/ml sodium fluoride solution (Sigma) and the OD measured at wavelength 630 nm with a micro-ELISA plate reader (Dynatech, MR 600).
Statistical analysis. Data were analyzed using Student's t test and coefficient of correlation (r).