Sandwich-type immunoassay with an affinity column coupled to anti-peptide antibodies

A new sandwich-type immunoassay method utilizing an anti-peptide antibody column and a fluorescence-labeled antibody (FLA) was proposed and applied to the measurement of the concentrations of insulin and insulin B-chain. In this method an immunoadsorbent coupled with an anti-peptide antibody was packed in a small column. A target protein was adsorbed on the anti-peptide antibody in the column, coupled with the FLA as a secondary antibody in sandwich manner and then eluted by an eluent containing an antigen peptide to the anti-peptide antibody. The concentration of the target protein was determined form the fluorescent intensity of the eluted FLA. It was possible to determine the concentrations within 30 min, and the higher sensitivity was obtained in comparison with the direct method using an immunoaffinity column.