determination of serum cortisol level by a direc t enzyme immunoassay using penicillinase as label

serum cortisol level was measured by an enzyme immunoassay (eia) using the enzyme penicillinase as a label without prior extraction and purification. polyclonal antibodies were raised against cortisol-3-ortho-carboxymethyl-oxime (cortisol-3-0-cmo) conjugated to bovine serum albumin (bsa). this antibody showed a very low cross-reactivity with structurally related steroids (3.7% for corticosterone and 4% for ii-deoxycorticosterone). standard doses were prepared in a serum sample stripped from endogenous cortisol. danazol, 8-anilinonaphtalosulphonic acid (8-ans) and salicylic acid were used as blocking reagents. however these reagents were not suitable in this assay. samples were heat treated (60° c for 30 min) in order to denature the binding proteins. the assay was sensitive from 250 pg per tube covering up to 50 ng with each point having a coefficient of variation (cv) of less then 15% throughout ten successive assays. cortisoi3-0-cm 0 was conjugated to the penicillinase following a carbodiimide procedure. the formed conjugate retained almost 90% of the enzyme activity. recoveries of exogenously added cortisol from charcoal-stripped plasma in three different ranges varied between 90-100%. inter and intra-assay variations showed a cv of less than 12 %. the correlation coefficient was calculated as r=0.99 using our method and the results reported by a local hospital for 20 samples.