Abstract
An immunoassay based on a monoclonal antibody for the analysis of adrenocorticotropic hormone (ACTH) in human serum has been developed. The principle of this immunoassay is the competition between human ACTH and ACTH-EGFP (Enhanced Green Fluorescent Protein) fusion protein. We constructed a prokaryotic vector expressing ACTH-EGFP gene and purified this His˙tag fusion protein from Escherichia coli cultures using nickel resin. Surface plasmon resonance (SPR) was used as an independent means of determining the affinity of ACTH-EGFP, human ACTH 1-39 and anti-ACTH monoclonal, polyclonal antibodies. The interaction between anti-ACTH monoclonal antibody and ACTH-EGFP, Human ACTH 1-39 was demonstrated to be of high affinity (1.94×10-12 M and 1.36×10-8 M, respectively). Finally, human ACTH concentration determined by this competitive enzyme-linked immunosorbent assay (CELISA) ranged from 4~10 ng/ml.