Biomedical subjects

Oliver J Kreuzer

Publications and source records attributed to Oliver J Kreuzer.

3 recordsLinked to original sources

PubMed2006

Agonist-induced endocytosis of rat somatostatin receptor 1.

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand.

Acids↗

PubMed2006

Development of peptide microarrays for epitope mapping of antibodies against the human TSH receptor.

Accurate characterization of the antigen binding region of antibodies is of great value in many fields of research, assay development and clinical diagnostics. Up to now, there is an unmet clinical need to use antibodies as diagnostic markers for the prediction of both prognosis and therapeutic response. To this end, comprehensive but differentiated immunoassays need to be generated. We have developed a peptide microarray for the diagnosis and epitope mapping of anti-thyrotropin receptor antibodies. The primary sequence of the human thyrotropin receptor (hTSHR) was represented by a library of 251 synthetic peptides. The peptides were site-specifically immobilized in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde coated glass surface. The technology was used to map the epitopes of seven commercially available murine monoclonal antibodies specific for the human TSH receptor (mTSHRAb). A previously unknown epitope recognized by mTSHRAb 4C1 was identified at amino acids (AA) 379 through 384 and the epitope recognized by mTSHRAb A9 was also localized (AA 214-222). Previously identified epitopes recognized by mTSHRAbs 2C11 (AA 349-360), 28 (AA 34-39), 49 (AA 289-297), A7 (AA 406-411) and A10 (AA 34-39) were confirmed. The peptide microarray exhibited excellent performance in single and multiplex antibody analysis and high specificity. This technology may have potential as a multi-determinate in vitro diagnostic assay for the differential analysis of a heterogeneity of antibodies involved in the pathogenesis of autoimmune diseases.

Aldehydes↗

PubMed2006

Functional peptide microarrays for specific and sensitive antibody diagnostics.

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.

Amino Acid Sequence↗