<p>Proteins exist in a variety of isoforms with minor differences, mostly due to their glycosylation patterns, which can modulate their biological functions. It seems to be of clinical relevance to measure the isoform-distribution.</p><p>Thesis describes a novel technology named Membrane-Assisted Isoform ImmunoAssay (MAIIA). This technique allows rapid (< 15 min.) isoform determination. It is based on a chromatographic separation combined with immunoassay detection. These steps are performed along a thin, disposable micro-porous chip in which capillary forces maintain the flow. By using anion-exchange as a chromatographic principle the technology has been utilized for the determination of transferrin isoforms in ten minutes. In one variant (the one-dimensional), selected isoforms (carbohydrate-deficient transferrin) are quantified. In a more elaborate variant (the two-dimensional) it was possible to determine the entire isoform profile of transferrin. Isoforms differing by only 0.1 pH unit in isoelectric point could be distinguished.</p><p>The chromatography along the microporous bed of nitrocellulose showed very good separation performance with plate heights of 10-20 µm and only minor flow rate variations between individual devices. </p><p>The quantitative determination of antibody-captured molecules was performed by using antibodies labelled with carbon black particles. Combined with a detection procedure by means of a flatbed scanner, a highly sensitive and specific immunoassay with a detection limit of 0.13 pM was obtained upon using IgE as a model analyte.</p><p>This technology can thus be used to rapidly distinguish proteins with minor structure differences and specifically determine protein isoforms in complex environments, e.g., blood, down in the pM (10<sup>-12</sup> M) concentration range.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:uu-1861 |
| Date | January 2002 |
| Creators | Lönnberg, Maria |
| Publisher | Uppsala University, Surface Biotechnology, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
| Relation | Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1104-232X ; 691 |
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