Preparation of an immunoadsorbent interacting specifically with cortisol-binding globulin (CBG) and its application to the interaction between CBG and cortisol--evaluation of the association constant (Ka) between CBG and cortisol in normal subjects

Abstract

We have developed a simple method for the evaluation of the association constant (Ka) between cortisol binding globulin (CBG) and cortisol using immunoadsorbent and a small amount of serum. Immunoadsorbent was prepared as follows; gamma-globulin fraction was obtained from goat anti-human CBG antiserum by salting out using 50% ammonium sulfate, followed by coupling to CNBr-activated Sepharose 4B. Immunoadsorbent thus prepared was used for the evaluation of Ka value between cortisol and CBG. Sera obtained from 5 healthy subjects (3 males and 2 females) were treated with charcoal (Norit A) to remove endogenous cortisol. Each of 10 microliter of serum was incubated with a constant amount of 125I-cortisol and different concentrations of unlabelled cortisol for 12 hours at 25 degrees C, pH 7.4. Then, 50 microliter of anti-CBG-Sepharose 4B (immunoadsorbent) was added to each incubation mixture, followed by incubation for another 4 hours at 25 degrees C. After centrifugation, radioactivity of the precipitate was counted and the results were analyzed using Scatchard plot. The Ka value between serum CBG and cortisol in 5 healthy subjects was 1.70 X 10(8)M-1 (SD = 0.24), which is comparable with that of a previously reported value using purified CBG from a large amount of serum. Interassay variation of this method was satisfactory with CV value of 10.1%. Our present method is considered to be clinically relevant, since it does not require purification of CBG from serum, and a relatively small amount (total amount, less than 200 microliter) of serum is enough to evaluate Ka value between CBG and cortisol.