Membrane assisted isoform immunoassay. A rapid method for the separation and determination of protein isoforms in an integrated immunoassay.

Proteins often exist as isoforms with structural microheterogenity due to, for example, small variations in their carbohydrate structure. This can give rise to differences in biological activity. Measurements of low concentrations of such isoforms are usually performed by complicated methods, which demand discrete steps of separation (chromatographic or electrophoretic) and immunodetection. In this paper a new, highly specific and rapid (<15 min) analytical technique is described which permits the quantitative determination of several types of protein isoform. The method, which is called membrane assisted isoform immunoassay (MAIIA), is based on separation (by ion-exchange or affinity chromatography) and immunoassay detection of the protein isoforms in the same lateral flow, assisted by capillary forces in a membrane device. The technique is exemplified with a method for measuring carbohydrate-deficient isoforms of the glycoprotein transferrin, where the measured isoforms constitute a minimal part (<3%) of the total amount of transferrin. The time for the measurement was about 10 min and the correlation coefficient with an established, commercial two-step procedure, which takes 4-5 h to perform, was 0. 99. The detection limit was about 1 fmol of transferrin. The anion-exchange membrane used in the test device had the ability to separate transferrin isoforms with down to 0.1 unit difference in pI. The results indicate that the technique should be useful for rapid point-of-care testing of clinically interesting charged protein isoforms.