Membrane-Assisted Isoform ImmunoAssay : Separation and determination of protein isoforms

Sammanfattning: 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.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.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. 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.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 (10-12 M) concentration range.