The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion.
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.
Lindbladh
C
C
Pure and Applied Biochemistry, Chemical Center, University of Lund, Sweden.
Persson
M
M
Bülow
L
L
Stahl
S
S
Mosbach
K
K
D000469:Alkaline Phosphatase / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007328:Insulin / Q000032:analysis*; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins
D007328:Insulin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011384:Proinsulin; D000469:Alkaline Phosphatase