New applications of carbonylmetalloimmunoassay (CMIA): a non-radioisotopic approach to cortisol assay.

A non-isotopic heterogeneous competitive immunoassay procedure, carbonylmetalloimmunoassay (CMIA) has been applied to the assay of cortisol. The organometallic tracers employed were two stereoisomers (Z and E) of certain cobalt carbonyl complexes of cortisol which have strong, characteristic v(CO) absorptions in the infrared, detectable by Fourier transform infrared (FT-IR) spectroscopy at the femtomole level. The separation of the free and bound organometallic-labelled fractions was achieved by solvent extraction with isopropyl ether. Complete characterization (i.e., dilution, competition and standard curves) of two different polyclonal anticortisol antibodies was possible using the CMIA method. Identical titre values were obtained for the two different stereoisomers used as tracers. In terms of the standard curves, however, the isomers behaved differently depending on which batch of antibody was used. When the best antibody/tracer pair (30 pmol of E isomer; anticortisol 1 antibody) was employed, we obtained a B/B(o)value at 50% of 42 +/- 2.12 pmol and a coefficient of variation of 5%. Finally, preliminary results of a CMIA analysis of plasma cortisol from a patient indicated that reliable and reproducible assays are possible for amounts as small as 50 microliters of serum.