Sensors and Actuators B 45 (1997) 63–69 Analysis of cortisol with a flow displacement immunoassay Wilhelmina A. Kaptein a,b, *, Jacco J. Zwaagstra a , Kor Venema a , Marcel H.J. Ruiters b , Jakob Korf a a Department of Biological Psychiatry, Uniersity of Groningen, Hanzeplein 1, 9713 EZ Groningen, The Netherlands b Biomedical Technology Centre, Uniersity of Groningen, Hanzeplein 1, 9713 EZ Groningen, The Netherlands Received 27 November 1996; received in revised form 25 August 1997; accepted 28 August 1997 Abstract This study investigates a flow immunoassay for cortisol based on displacement of labelled antigen analogues from immobilised antibodies. On a displacement column, two antibodies with different affinities were immobilised on protein-A Sepharose, and a construct of cortisol and horseradish peroxidase (cort-HRP) was associated to the antibodies. Perfusion of the column with cortisol displaced a proportional amount of cort-HRP. On-line detection was performed with an electrochemical detector measuring cort-HRP directly in a flow injection system. For investigating some characteristics of the displacement system, cort-HRP was also detected off-line, with a substrate for HRP, which changes OD 492 . We tested our system in buffer as well in serum for 2 days at a flow of 4–6 l min -1 . Analysis of the data show a detectable displacement signal at physiological cortisol concentrations. The specificity, stability of the columns and the influence of the antibody affinity were studied. © 1997 Elsevier Science S.A. Keywords: Immunosensor; Cortisol; Displacement; Analyte-enzyme conjugate; Electrochemical detection 1. Introduction Total serum cortisol concentrations are 20 – 200 g l -1 (55–550 nM) of which over 90% is bound to proteins and has daily fluctuations. To get accurate measurements of the amount of cortisol or of the circadian profile, one needs to measure blood several times a day. Besides this, for some clinical diagnosis, it is necessary to know the exact cortisol fluctuations during the day [1]. To comply with this, it is necessary to develop a technique, which enables analysis of con- tinuous samples. There are several methods to measure cortisol in body fluids. The conventional method, analysis of sera with a HPLC is very labour intensive [2]. Radio im- munoassays have been used for some time now [3], but they have the disadvantage of using radioactive com- pounds. New techniques, using antibodies and labelled corti- sol have been developed during the last decade [4]. Nowadays, there are some fully-automated analysers available for the analysis of cortisol, such as Enzymun- Test Assay (Boehringer), ACS:180 (Ciba Corning Diag- nostics) [5] and Stratus (Baxter) [6]. Most of these are based on competitive antibody-antigen assays, where immobilised antibodies are incubated simultaneously with a sample and a known amount of enzyme-labelled cortisol. The cortisol concentration is, therefore, in- versely proportional to the enzyme activity. Also, a piezoelectric immunosensor [7] and capillary elec- trophoresis method [8] for cortisol have been studied recently. However, none of these methods allow the continuous monitoring of cortisol in vivo. For this, a homogeneous assay is essential. One suitable technique for on-line analysis is a displacement system, described by Kusterbeck, Rabbany et al., e.g. for cocaine and dinitrophenol [9 – 13]. Here, antibodies are covalently Abbreiations: cort-HRP, cortisol conjugated to horseradish perox- idase; ECD, electrochemical detection; M cort-H, a high affinity monoclonal antibody against cortisol; M cort-L, a low affinity mon- oclonal antibody against cortisol; SB, special buffer for displacement experiments; GB, a glucose containing buffer; OPD, o -phenylene diamine dihydrochloride; ABS, antibody binding site. * Corresponding author, Department of Biological Psychiatry, University Hospital AZ/RUG P.O.Box 30.001, NL 9700 RB Gronin- gen, The Netherlands. Tel.: +31 50 3612099; fax: +31 50 3611699; e-mail: W.A.Kaptein@MED.RUG.NL 0925-4005/97/$17.00 © 1997 Elsevier Science S.A. All rights reserved. PII S09 2 5 -4 005(97)00 2 7 1-2