Enzyme Immunoassay of Adrenocorticotropic Hormone-like Immunoreactive Substance in Human Plasma

A sensitive and specific double-antibody enzyme immunoassay (EIA) for adrenocorticotropic hormone-like immunoreactive substances (ACTH-ISs) was developed. In competitive reactions, the ACTH (1-24)-antibody was incubated with both ACTH (1-24) standard (or sample) and 13-D-galactosidase labeled synthetic human ACTH (1-24) (delayed addition). The free and antibodybound enzyme haptens were separated by using an anti-rabbit IgG coated immunoplate. The activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows for the detection of 2 to 600 fmol/mL of ACTH (1-24). Using the proposed EIA, ACTH (1-24)-ISs in human plasma were thus determined. As a result, the levels of ACTH (1-24)-ISs in human plasma were found to be about 3.7 fmol/mL.