Engineering of a Recombinant Colorimetric Fusion Protein for Immunodiagnosis of Insulin

A synthetic DNA encoding human proinsulin was inserted in frame in the bacterial alkaline phosphatase gene. A homogeneous recombinant human proinsulin-alkaline phosphatase conjugate was obtained directly from the periplasm of Escherichia coli transformed with a plasmid carrying the hybrid gene. The recombinant conjugate was stable and could be produced in the bacteria. The immunological properties of the recombinant conjugate and those of the human insulin and human proinsulin were compared using a panel of six different human insulin-specific monoclonal antibodies. Three immunological groups were thus distinguished and one of them indiscriminately recognized all of the insulin-like molecules. One monoclonal antibody from this group was used in combination with the recombinant conjugate to develop successfully a competitive immunoenzymatic assay for detecting insulin.