The monitoring of COMP levels in synovial fluid or serum has been suggested to be a helpful method for assessing the presence and progression of arthritis. well correlated with VU6005806 severity in OA patients and the progression of surgically induced OA in murine models. Furthermore, the serum levels of COMP fragments in RA patients, mice with CIA, and TNF transgenic mice were significantly higher when compared with their controls. Interestingly, treatment with TNF inhibitors and methotrexate led to a significant decrease of serum COMP fragments in RA patients. Additionally, administration of Atsttrin (Tang, et al, gggcgacaggccgcga-3), forward primer P3 (5-aggatccand sites are underlined and the italic letters represent three parts of the signal peptide sequence of COMP. After 3 cycles, these sequences were joined together to generate the intact N-terminal signal peptide, which facilitated the recombinant protein secretion into the culture medium. The amplified DNA fragment was ligated into the and sites of pCDNA3.1-myc-his A (Invitrogen). Expression and purification of recombinant COMP type III in HEK 293EBNA cells HEK 293EBNA cells were transfected with 20 g DNA using Lipofectamine 2000 (Invitrogen). The stable lines were generated by G418 (Sigma) selection. The serum-free conditioned medium VU6005806 was mixed with Probond? Nickel-Chelating Resin (Invitrogen). The bound proteins in the washed resin were eluted with 250 mM Imidazole. The secretion of recombinant protein was confirmed by SDS-PAGE. Additionally, the recombinant proteins were analyzed by Western blotting using a rabbit anti-COMP polyclonal antiserum (pAb) or anti-COMP type III monoclonal antibody (mAb) 2127F5, respectively. Generation and partial characterization of mAbs against the COMP type III domain name Female Balb/c mice were immunized with the recombinant type III domain name of COMP (aa 266C526) and hybridomas were produced according to standard procedures. Procedures for spleen fusion and clone selection were as previously described[25]. Recombinant human COMP was incubated with MMP13 catalytic domain name (Abcam) or purified catalytic domain name of ADAMTS-7 in digestion buffer (50 mM Tris-HCl, 100 mM NaCl, VU6005806 5 mM CaCl2, 2 mM ZnCl2, and 0.05% Brij-35, pH 7.5, substrate and enzyme molar ratio 6:1) at 37C for 16 hrs. The digested products were analyzed by Western blotting using selected mAbs or the pAb against COMP. Epitope mapping of anti-COMP mAb 2127F5 An overlapping peptide library for human COMP type III domain name was designed and generated by Genscript Inc (Piscataway, New Jersey). Each peptide of the library was 15 amino acid long with 10 amino acids overlapping with its adjacent peptide. All cysteines in the peptides were replaced with serine. A total of 51 biotinylated overlapping peptides were synthesized and screened by ELISA with mAb 2127F5. Immunoblotting for RH-II/GuB COMP in serum A volume of 10 l of hyaluronidase (100 U in 0.05 M sodium acetate buffer, pH 5.8) from bovine testis (Sigma) was added to 50 l of serum from normal control, OA patients, and RA patients. After incubation at 37C for 2 hrs, samples were precipitated by adding 1 volume of 100% Trichloroacetic acid to 4 volumes of serum, and incubated on ice for 20 min. The supernatant was spun and the pellet VU6005806 washed first with ice-cold acetone twice and then dissolved in non-reducing SDS-PAGE loading buffer. Soluble material was subjected to SDS-PAGE and detected with either the anti-COMP pAb or mAb 2127F5. Immunohistochemistry Specimens of articular cartilage from OA and RA patients and normal controls were fixed in 4% PBS buffered PFA at 4C overnight and then decalcified in 10% EDTA for 4 weeks. After blocking, tissue sections were incubated with mAb 2127F5 at 4C overnight, followed by incubation with a biotinylated secondary anti-mouse IgG for 1 hr at room temperature. Following intermittent rinses in PBS, avidin-biotin-peroxidase complex (Vector Elite ABC Kit; Vectastain) was applied for 1 hr and signal detection was performed by using diaminobenzidine for 10 min (Sigma-Aldrich) as the substrate, followed by a counterstaining with methyl green. Sandwich ELISA Briefly, the anti-COMP pAb[26] was used as a capture antibody, whereas.