Membrane vesicles, bound to SF proteins in a calcium-dependent manner, were washed twice using this buffer in order to eliminate unspecifically bound proteins. The
specifically bound proteins were released from membrane by including 1 mM EGTA minus calcium-containing buffer by centrifugation at 28 000 g for 30 min at 4°C. The supernatant containing NAP was dialysed and purified further by size exclusion chromatography using Sephadex G-100, after which its identity was determined by peptide mass fingerprinting and N-terminal protein sequencing. The purified fraction was assayed for proangiogenic activity using human umbilical vein endothelial click here cells mTOR inhibitor (HUVECs) for tube
formation . Purified NAP was used to produce monoclonal antibody. Briefly, BALB/c mice were immunized four times over a 2-month period with 50 μg of purified NAP with Freund’s adjuvant. Serum samples were collected 2 weeks after the second, third and fourth immunizations and screened for anti-NAP antibody using indirect ELISA. Spleen from mice that displayed high antibody titres were used subsequently to generate hybridomas using standard spleen cell/myeloma fusion. Briefly, NAP-primed B cell 1 × 108 (splenocytes) from mouse producing high-titre neutralizing antibodies were fused with logarithmically growing Sp2/0 myeloma cells (1 × 107), using polyethyleneglycol-1500. Hybridoma selection was carried out in hypoxanthine–aminopterin–thymidine (HAT) medium. The resulting monoclonal hybridomas were grown to confluency and the cell supernatant from a single clone was collected as a source of anti-NAP mAb, verified using
ELISA in which NAP was used for capture of the anti-NAP mAb, and purified by protein-A agarose affinity column chromatography. Further immunodetection Urocanase of anti-NAP mAb was carried out by Western blot analysis. Arthritis was induced in Wistar rats by subcutaneous (s.c.) injection of NAP or ovalbumin (OVA; Sigma, St Louis, MO, USA), as described previously . There were five groups containing six animals, each in duplicate, as follows: group 1, controls; group 2, positive control [OVA-induced arthritis (AIA; untreated)]; group 3, NIA untreated; groups 4 and 5 served as test (AIA DMRD-treated and NIA mAb-treated), respectively. All rats except controls were sensitized twice during a 6-week period with 2 mg/ml of OVA or 50 μg/ml NAP emulsified in complete Freund’s adjuvant (CFA) (Sigma) and administered s.c. At the end of 6 weeks, animals received an intra-articular injection of 2 mg/ml of OVA or 50 μg/ml NAP in CFA in order to induce arthritis. The control rats were injected only with Freund’s adjuvant. Arthritis was achieved in 6–7 days post-IA injections and was considered as day ‘0’.