The design of a simple competitive elisa using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion

Abstract

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene ( pho A ) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5′-terminal end of pho A . In the other construction a 42 base pair DNA fragment has been deleted from the 3′-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10–15 U/mg and 18–25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.