The standardization and clinical validation of the measurement of beta-amyloid(1-42) (Abeta42) in cerebrospinal fluid (CSF), plasma and urine is described using a commercially available sandwich-type ELISA with 21F12 and 3D6 as monoclonal antibodies. The INNOTEST beta-amyloid(1-42) allows the specific and reliable measurement of(1-42) amyloid peptides in CSF and plasma. The Abeta42 concentrations in serum and urine were below the detection limit. In plasma, no differences were found in Abeta42 levels between controls and patients with different neurodegenerative disorders (Alzheimer's disease (AD), Lewy body disease (LBD), others). In contrast, CSF-Abeta42 concentrations were lower in AD and LBD patients as compared to controls. No correlation was found in AD patients between CSF and plasma concentrations of Abeta42 or between CSF Abeta42 levels and blood-brain-barrier function. The quantitative outcome of the test is in part dependent on confounding factors such as tube type, freeze/thaw cycles, temperature of incubation, standard preparation protocol, and antibody selection. Notwithstanding these aspects, it emerged that Abeta42 is a useful biochemical marker for the diagnosis of AD patients, but there is a need for an international Abeta standard, a universally accepted protocol for CSF preparation, and a thorough evaluation of assay performance in function of the boundary conditions.

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Purpose: Healthy cartilage homeostasis depends on an intact collagen scaffold and high aggrecan content. ADAMTS-5 (A Disintegrin And Metalloprotease with ThromboSpondin-motifs-5) is critically involved in arthritic diseases because of its direct role in cleaving aggrecan. Several studies indicate that inhibition of ADAMTS-5 may have the potential to stop progression of osteoarthritis (OA). In the present study, we investigated the in vitro efficacy of M6495, an inhibitor of ADAMTS-5, including assessment of affinity, potency, specificity, and its effect on the levels of glycosaminoglycan (GAG) and the ADAMTS-5 generated neoepitope of aggrecan (huARGS) in bovine and human cartilage explant assays. Furthermore, we studied the effect of M6495 on cartilage derived markers from aggrecan (GAG) and type II collagen (C2M), as well as type III collagen (C3M) in a cartilage-synovium co-culture model. Methods: Binding kinetics of M6495: The affinity of M6495 for human ADAMTS-5 was determined via Sapidyne's 'in solution' affinity platform Kinetic Exclusion Assay (KinExA). To determine the binding region of M6495 within ADAMTS-5, binding experiments were performed using SPR (Surface plasmon resonance). Inhibition of enzymatic activity of ADAMTS-5 by M6495 was analyzed with a fluorescence resonance energy transfer (FRET)-based enzymatic activity assay. The specificity of M6495 towards ADAMTS-5 was assessed by binding experiments to homologous metalloproteinases. Explant assay and co-culture: The effect of M6495 (half maximal inhibitory concentration [IC 50 ]) in bovine and human cartilage explants stimulated with pro-inflammatory cytokines was determined. Biomarkers of cartilage turnover, including GAG and huARGS, were investigated in the supernatant after culture had ended. Bovine cartilage explants and synovial membranes were coincubated. GAG as a measure for aggrecan turnover, C2M as a marker for MMP (matrix metalloproteinase) mediated type II collagen degradation and C3M as a marker for MMP-mediated type III collagen degradation were analyzed in the supernatant of the cultures. Results: M6495 is a bifunctional Nanobody® of 28.1 kDa (i.e., one ADAMTS-5-neutralizing moiety and one [HSA]-binding moiety for in vivo half-life extension). M6495 binds ADAMTS-5, but not ADAMTS-1, ADAMTS-4 and ADAMTS-15. In-vitro binding studies revealed that M6495 binds to the catalytic and/or disintegrin domain of ADAMTS-5 with high affinity. Results from FRET assays indicate a concentrationdependent and complete inhibition of the enzymatic activity of ADAMTS-5 by M6495. M6495 dose-dependently inhibited GAG release in bovine-, and GAG and huARGS release in human cartilage explant assays. In the co-culture model, matrix degradation was induced in the presence of synovium. First aggrecan was degraded (GAG release) followed by type II collagen degradation (C2M release). M6495 inhibited both. In addition, C3M was determined in the supernatant of the coculture system. Our data revealed a significant induction of C3M levels in co-cultures compared to t...

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