Methods: The BC3 monoclonal antibody that detects the ARCS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis find more to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized ct-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial
fluid, serum and urine were detected and quantified
using this ELISA.
Results: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARCS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARCS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARCS containing fragments www.selleckchem.com/products/jq1.html in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity.
Conclusions: We have developed a novel ELISA using an optimized ARCS antibody and have demonstrated for the first time, an ELISA-based EPZ-6438 research buy measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker
in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA. (C) 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.”
“Objective: The goal of this prospective study is to investigate the correlation between allergen sensitivity and radiographic evaluation of adenoidal obstruction and tonsil size.
Subjects and Methods: A total of 82 children with upper airway obstructive symptoms were evaluated for their tonsil size. All patients underwent digital lateral soft tissue radiographs. Assessment of nasopharyngeal obstruction in radiographs was done according to the Cohen and Konak method. Skin prick tests with multitest applicator including 14 allergens were carried out to investigate their allergic background.