After 24 h of culture, the CTLL cells were pulsed with [3H]thymidine for an additional 4 h and the net cpm (mean±SD) selleck screening library were calculated. HLA-DR2 mice between 8 and 12 wk of age were immunized s.c. at four sites on the flanks with 0.2 mL of an emulsion of 200 μg mouse MOG-35-55 peptide and complete Freund’s adjuvant containing 400 μg

of Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA). In addition, mice were given Ptx from List Biological Laboratories (Campbell, CA, USA) on days 0 and 2 post immunization (75 and 200 ng per mouse, respectively). HLA-DR2 mice were treated with vehicle, RTL342m alone, or RTL342m pre-incubated with one of the FAbs beginning on the first day that the combined clinical EAE score for each individual mouse reached

2 or higher. Once-daily treatments were administered to each mouse subcutaneously in the interscapular region for three days. RTL342m and RTL342m+FAb were prepared in Z-VAD-FMK 100 μL of 20 mM Tris-HCl pH 8.0 with 5% w/v D-glucose (Sigma-Aldrich, St. Louis, MO, USA). Vehicle treatments consisted of only Tris-HCl pH 8.0 with 5% w/v D-glucose. Mean EAE scores and SDs for mice grouped according to initiation of RTL or vehicle treatment were calculated for each day. The CDI was determined for each mouse by summing the daily EAE scores. Group CDI scores were calculated by determining the mean±SD of the individual mice in the group. The IACUC Protocol ♯2108, Vandenbark AA PI, was in

place and is currently approved for the animal experiments reported in the manuscript. Detection of RTL-like material in human serum or plasma was determined by ELISA using Fab 1B11. ELISA plates (Falcon) were coated for 2 h with anti-MHC mAb TU39 (10 μg/well). The plates were blocked for 30 min at room temperature with PBS/2% skim milk CYTH4 and subsequently were incubated for 2 h at room temperature with serial dilutions of RTL1000 (for standard curve) and 1:10 serum dilutions. After being washed, the plates were incubated (1 h at room temperature) with 1B11 Fab (10 μg/mL), washed extensively and further incubated (1 h at room temperature) with anti-myc-biotin Ab (9E10 clone, Covance). The plates were washed and incubated for 30 min with HRP-conjugated streptavidin. Further amplification steps were performed using the ELAST ELISA amplification system (PerkinElmer), according to the manufacturer’s protocol. Detection was performed using TMB reagent (Sigma). Detection of RTL1000 in human serum or plasma was determined by ELISA using biotinylated Fab 2E4. ELISA plates (Falcon) were coated overnight with BSA-biotin (1 μg/well). After being washed, the plates were incubated (1 h at room temperature) with streptavidin (10 μg/mL), washed extensively and further incubated (1 h at room temperature) with 5 μg/mL of biotinylated Fab 2E4.