Volume 11, Number 4 (2-1998)                   Med J Islam Repub Iran 1998 | Back to browse issues page


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POUR-AMIR M, RASAEE M, DJALALI M, MALEKANEH M. DETERMINATION OF SERUM CORTISOL LEVEL BY A DIREC T ENZYME IMMUNOASSAY USING PENICILLINASE AS LABEL. Med J Islam Repub Iran. 1998; 11 (4) :335-340
URL: http://mjiri.iums.ac.ir/article-1-1078-en.html

From the Biochemistry Department, Tarbiat Modarres University, School of Medical Sciences, Tehran, I.R.lran
Abstract:   (2145 Views)
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.
Keywords: EIA, Cortisol, Penicillinase
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