Affinity-arrays in clinical diagnostics

Abstract

Microarray technology makes the highly paralleled analysis of multiple simultaneous reactions possible. This study is centred on issues of identification of protein biomarkers present in serum/plasma at low concentrations, parameter optimization in protein array production and assaying as well as a novel type of diagnostic microarray device suited for early-on disease diagnosis via auto-antibodies.<br />First, the effectivity of the depletion of human high abundance serum and plasma proteins for improved protein identification and the risk of concomitant removal of relevant marker proteins were assessed. 2-D gel electrophoresis and shotgun mass spectrometry combining 2-D capillary chromatography with MS/MS were applied in parallel for the analysis of the fractions. Unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients was detected. Protein microarray substrates were scrutinized for the use as protein antigen and antibody microarray supports. For the assessed microarray substrates with 2-D and 3-D surface chemistries, ideal candidates could be identified delivering highly reproducible and stable data. A range of methods of detection on antibody arrays has been compared and evaluated.<br />For the detection of auto-antibodies for early-on disease diagnosis, macro-arrays comprising 38,000 clones of E. coli expressing proteins of a fetal brain library were probed with patients' and control sera.<br />Results of these screens were proceeded on to assay miniaturisation.<br />Recombinant expression in multi-well plates was established together with protein purification procedures. Conditions of microarray processing and biomarker detection were successfully optimized.<br />Microarray binding reactions with patient's sera reached highly reproducible signal patterns.