A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

Preparation and validation of implantable electrodes for the measurement of oxygen and glucose.

In realizing the continuous measurement of pO2 and glucose concentration in the subcutaneous tissue, miniaturized electrochemical oxygen- and enzyme glucose sensors, based on modified Clark-type electrodes for transient implantation, were developed and investigated. The electrodes were prepared by means of sequential dipping procedures in glucose oxidase and in different polymer solutions at well-defined environmental conditions in an incubator. By means of combining a hydrophobic membrane with a glucose permeable area and a hydrophilic membrane in the case of the glucose sensor, linearity of the glucose dependent electrode signal up to greater than 20 mmol/l could be achieved. After subcutaneous implantation in the neck of dogs, the enzyme sensor is able to follow glucose profiles in the normo- and in the hyperglycaemic range, e.g. as caused by oral glucose loads. Looking for the difference in sensitivity of the enzyme sensor measured in vitro and calculated from in vivo data, the influence of potential nonspecific, interfering substances in vivo such as urea, amino acids, electrolytes, and albumin was estimated.