Paws were thickened in mice when only sera were transferred. MC-Val-Cit-PAB-rifabutin 50g/ml. The cells were incubated for 48 h at 37 C. During the last 18 h the cells were pulsed with 3H-thymidine (1Ci/well). At the end of the assay, the cells were harvested using a plate harvester and integrated radioactivity was identified using an automated counter (Micrebeta, Perkin Elmer Wallac). Peptides derived from human being type II collagen (HII) were synthesized and purified at Mayo Medical center Peptide Facility. The mice were primed with 200 g of peptide emulsified 1:1 with CFA and challenged with 100 g/ml of the peptide. To determine the restriction molecule for the in vitro response, obstructing studies using anti HLA-DQ antibodies were performed. HII derived peptide 554?573 was used to assess the demonstration of antigen by sorted B cells and dendritic cells to T cell hybridoma specific for the peptide. Also, B and DCs MC-Val-Cit-PAB-rifabutin were incubated with peptides HII-44 and 44S for 24 hrs and then washed and cultured in the presence of hybridomas without the peptides. Peptide ELISA For B cell epitopes, sera from CII and PBS immunized mice were analyzed for reactivity to CII-derived peptides by ELISA. Peptides utilized were of various cyanogen bromide (CB) fragments of Human being type II collagen (HII) and displayed these sequences; CB6 fragment (F=44?63, G=54?73, K=94?113) CB11 fragment, (7=184?203, 17=184?303) CB8 fragment (33=444?463, 38=494?513) CB10 fragment (43=544?563, 44=554?573, 48=594?613) and CB9/7 fragment (61=724?743, 64=754?773). Briefly, Maxisorp (NUNC) plates were coated with peptides at a concentration of 10 g/ml in 0.1M NaHCO3 buffer having a reaction MC-Val-Cit-PAB-rifabutin volume of 100l at 4o C overnight overnight. The non-specific sites were clogged 300ul of 3%BSA for 1hr at space temperature. Duplicate set of sera were used at a dilution of 1 1:100 and incubated at 4o C over night. Plates were washed 3 times with 0.5%Tween 20 and incubated for one hr at room temperature with 100ul of anti mouse Alkaline Phophatase (Jackson Immunoresearch). Plates were washed 5 occasions and developed using PNPP substrate tablets (Southern Biotech). Reaction was halted with 1M NaOH and plate read at 405nm. Cytokines Cytokines were measured in supernatants collected after 48 hrs from cultured splenic cells as explained above. Capture ELISA was carried out for measuring cytokines IFN and IL-4 using packages (BD Pharmingen, San Diego, CA). Cells isolated from 2?3 primed mice were cultured in vitro as explained above and supernatants were pooled. The experiment was repeated twice. Statistical Analysis Difference in incidence of arthritis between organizations was analyzed using chi-square test with Yates correction. Antibody levels and means scores for arthritic mice were compared using two-tailed College students test. Antigen presentations were analyzed using T test with unequal variance. Results Antibody reactivity to Collagen-derived peptides in DQ8 mice T cell epitope mapping of overlapping HII derived peptides showed at least 16 epitopes from numerous CB fragments of CII, which are DQ8-restricted (Krco et al, 1999). We tested if B cell response was much like T cell response using peptide ELISA. Known DQ8-restricted T cell epitopes were utilized. Even though B cell reactivity with the peptides was much like T cells with most of the peptides showing reactivity to B cells, the strength of reactivity differed. Some peptides which offered a strong response with T cells experienced milder antibody reactivity (Fig 1, 33). There was a strong antibody response to CII in sera from immunized mice. Open in a separate window Number 1 Peptide ELISA using sera from DQ8 mice showed that a differential B cell reactivity with known DQ8-restricted T cell epitopes. A) Sera from primed were tested on plates coated with known DQ8-restricted T cell epitopes of human being type Mouse monoclonal to NME1 II collagen-derived peptides. Peptides sequences are explained in methods. B) Sera from primed.