2.1. Computer search
The BLAST database searching program was used to search the PIR-Protein database (V.33.0) containing 42,215 sequences. The sequence QTDRED from HLA-B27 and K. pneumoniae were compared with all the sequences in the database allowing for mismatches.
Sera were collected from 97 active AS patients (New York criteria) (erythrocyte sedimentation rate (ESR) > 15 mm/h, all HLA-B27 positive) attending the AS Research Clinic at the Middlesex Hospital, and 25 active rheumatoid arthritis (RA) patients (ARA criteria) attending the Rheumatology Department at the Lister Hospital, Stevenage,
0014-5793/95/S9.50© 1995 Federation of European Biochemical Societies. All rights reserved. SSDI 0014-5793(95)00760-1
Herts. The London Blood Transfusion Service provided 25 healthy control sera. All sera were stored at -20°C until required.
2.3. Peptide synthesis and ELISA study
The synthetic peptides were prepared by solid phase synthesis, purity assays were performed by high performance liquid chromatography. All peptides had a purity of at least 90%. Three 16mer peptides were constructed: the B*2705 sequence (residues 67-83) CKAKAQTDRED-LRTLL, the pulD secretion protein sequence (residues 590-605) RPTVIRDRDEYRQASS, and a control peptide sequence made from a scrambled sequence of the pulD peptide, RFTVRSDIDYRQAESR. Sera were tested against the three peptides by non competitive enzyme linked immunosorbent assay (ELISA). The assay was carried out as follows: polystyrene microtitre plates (Dynatech) were coated with the synthetic peptide (5.0 ^g/well) overnight at 4°C. After absorption and washing with PBS-Tween, the plates were saturated with 2% casein-PBS-Tween, incubated for 1 h at 37°C, followed by washing with PBS-Tween. Serum samples (200 p\ at 1/50 dilution in PBS-Tween) were added to the plates, incubated for 90 min at room temperature, followed by washing with PBS-Tween and peroxidase conjugated rabbit anti-human class specific IgG, IgA or IgM (Dako. Ltd) diluted 1/500 in PBS-Tween added and plates incubated for 90 min at room temperature. After washing and enzyme reaction with substrate 2,2"-azinobis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS) (Sigma Chemical Co. Ltd.) at room temperature for 20 min, the reaction was stopped with sodium fluoride and the OD measured at wavelength of 630 nrn.
2.4. Organisms and growth conditions
Proteus mirabilis was obtained from the Department of Microbiology, King"s College whilst the Klebsiella pneumonias used was a clinical isolate kindly provided by the Medical Microbiology Department, Princess Royal Hospital, Haywards Heath. The cultures were grown aerobically in 250 ml conical flasks on an orbital shaker for 16-18 h in minimal media supplemented with 0.005% yeast extract (Difco) . If induction of the pullulanase enzyme system was required the starch substrate pullulan (0.3% w/v) (Sigma) was added to the minimal medium. The cells were harvested by centrifugation (MSB 18; 6 x 250 ml rotor) and washed in 0.15 M phosphate buffered saline (PBS). The suspension was then centrifuged as before. This procedure was repeated 3 times. After washing, the cells were resuspended in 20 ml of PBS and a stock solution was prepared to give an OD reading of 0.25 on the Spectrophotometer (Corning Model 258).
2.5. ELISA studies on the effect
of antibody binding to bacteria grown
in minimal media in the presence of pullulan
Aliquots of 200 (A of the diluted suspension of bacteria grown on minimal media plus 0.005% yeast extract (Difco) were adsorbed onto 96 well ELISA plates (Dynatech) overnight at 4°C. The ELISA procedure was carried out as described previously, with the exception of the application of the HRP-conjugated second antibody. In this assay, when K. pneumoniae was used as the antigen the HRP-conjugated second antibody was of the IgA class, whilst when Proteus mirabilis was
the antigen the HRP-conjugated antibody was of the IgG class. This experiment was repeated with a suspension of bacteria grown on minimal media plus 0.005% yeast extract (Difco) supplemented with pullulan (Sigma) (0.3% w/v).
2.6. ELISA studies on pullulanase (pulA)
Aliquots (200//I) of a solution of pullulanase (Sigma) (2//g/well) were adsorbed onto 96-well ELISA plates (Dynatech) overnight at 4°C. The ELISA procedure was carried out as described previously, however only IgA and IgG classes of the HRP-conjugated second antibody were used.
2.7. ELISA studies on collagen types I and IV
Solutions of collagen type I or type IV (Sigma) (3 /ig/ml) were adsorbed onto 96-well ELISA plates (Dynatech) overnight at 4°C in 200 fjl volumes. The ELISA plate was washed and satured with 2% casein-PBS-Tween described previously, however incubation for 1 h was performed at room temperature. Additionally, only IgA and IgG classes of the HRP-conjugated second antibody were used in this assay.