241
Clinica Chimica Acta, @ Elsevier/North-Holland
92 (1979)
241-247
Biomedical
Press
CCA 9986
FLUORESCENCE
POLARIZATION
YOSHIHARU KOBAYASHI KIYOSHI MIYAI b
a, KIYOKO
IMMUNOASSAY
AMITANI
a, FUKUKO
FOR CORTISOL
WATANABE
a** and
a Clinical Chemistry
Laboratory, Kobe Women’s College of Pharmacy, 4-19-1, Motoyama for Clinical Investigation, Kitamachi, Higashinada-ku, Kobe (Japan) and b Central Laboratory Osaka University Hospital, l-l-50, Fukushima, Fukushima-ku, Osaka (Japan)
(Received
August
31st, 1978)
Summary A fluorescence polarization immunoassay for serum cortisol was established using cortisol 21-amine which was readily coupled with fluorescein isothiocyanate. The proposed method is sufficiently sensitive, reliable, specific and simple for routine determination of serum cortisol. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of cortisol detected was 1.5 ng/tube and the measurable range was from 1.5 to 100 pg/dl. There was a good correlation between values obtained from radioimmunoassay and the proposed method.
Introduction Cortisol levels in body fluid have been measured mostly either by radioimmunoassay [l-3] or enzyme immunoassay [4-81. However, these assays are a time-consuming method to detect cortisol levels because of the requirement of antibody-bound/free separation, which is one of the restrictions on their application to autoanalysis. Radiation hazards and high cost of isotopes and enzyme are also disadvantages for these methods. In this paper we report a fluorescence polarization immunoassay for cortisol, which is a rapid method requiring no antibody-bound/free separation. The principle of this method is based on that described by Dandliker et al. [7]. However, the current method presents a novel approach to the measurement of haptens in vivo in practical use.
* To whom correspondence should be addressed.
242
Materials and methods Reagents
Cortisol 21-hemisuccinate was supplied by Japan Upjohn, Ltd. Cortisol and bovine y-globulin (BGG) were purchased from Sigma Chemical Co., U.S.A. Fluorescein isothiocyanate (FITC) was from Dojindo Lab., Japan. Thin-layer chromatograph plates (TLC) were obtained from Merck Co., Inc., U.S.A. Cortisol 21-amine was prepared according to the procedure described previously [6]. All reagent-grade organic solvents and reagents were used without further purification. Conjugation
reaction
of FITC
to cortisol21
-amine
All conjugation reaction was carried out away from daylight. A solution of 4 mg of cortisol 21-amine * HCl in 0.5 ml of methanol was adjusted to pH 9.0 with 0.1 M NaOH and mixed with 20 mg of FITC; finally, 0.4 ml of dioxane was added. The mixture was stirred at room temperature for 4 h. Then the reaction mixture was applied to TLC for purification. The developing solvent systems consist of ethylacetate/chloroform/methanol/acetic acid (5 : 3 . 1 : 1, v/v) and benzene/ethylacetate/ethanol (8 : 2 : 1, v/v). Antiserum
for fluorescence
polarization
immunoassay
for cortisol
Conjugation of cortisol 21-hemisuccinate to bovine serum albumin (BSA) was carried out by mixed anhydride reaction [8]. Anti-serum to cortisol 21-hemisuccinate-BSA produced in rabbits was prepared by Wako Pure Chemical Indust., Japan. Purification
of antiserum
Purified IgG of cortisol antiserum was prepared by applying to DEAESephadex A-50 column, which is commercially available as a kit “IgG TEST Chemiphar”, Nippon Chemiphar, Japan. As a stabilizer, 100 mg of BGG was added to 100 ml of the purified IgG solution. Measurement
of fluorescence
polarization
Fluorescence polarization was measured by a model IBF-129 polarization fluorimeter (Kowa Kizai Ltd., Japan). The excitation and emission wavelengths were 490 nm and 520 nm, respectively. During measurement of polarization, the temperature changes of cell chamber was controlled to within +0.5”C. Antibody
dilution
study
Purified anti-cortisol IgG in 0.01 M phosphate buffered saline solution (PBS), pH 7.4, containing 0.1% BGG was serially diluted with normal rabbit serum (NRS) IgG in 0.01 M PBS, pH 7.4, containing 0.1% BGG. Then 140 ~1 of methanol were added to 2 ml of fluorescein-labelled cortisol in 0.01 M PBS buffer, pH 7.4, and 40 ~1 of the diluted IgG solution were added to the assay tubes. The tubes were incubated at constant temperature from 25 to 30°C for 60 min. A fluorescence
To the
polarization
assay tubes,
immunoassay
procedure
100 ~1 of standard
or serum
samples
and 200 ~1 of
243
methanol were added. The tubes were centrifuged at 3000 rev./min for 20 min and 140 ~1 of methanol layer were transferred to the assay tubes. Then, 2 ml of FITC-cortisol solution in 0.01 M PBS, pH 7.4, was added and polarization (A) was measured by fluorescence polarization fluorimeter. Then, 40 ~1 of purified anti-cortisol IgG in 0.01 M PBS, pH 7.4, containing 0.1% BGG solution was added and the mixture was incubated at constant temperature from 25 to 30°C for 60 min. After the incubation, the polarization (B) was measured again. Interference of serum was corrected by subtracting the polarization value A from the polarization value B. All assays were done in duplicate. Radioimmunoassay
procedure
Cortisol radioimmunoassay was carried Immunochemical Institute, Tokyo.
out using a commercial
kit of Eiken
Results Conjugation
of FITC
Fluorescein-labelled
to cortisol21
cortisol
-amine
was found
to be pure from
TLC experiment.
0.
3.
l/240
l/960 antlbody
l/3840 flnal
l/15360
NRS/IgG
dllutlon
Fig. 1. Antibody dilution study. Purified anti-cortisol IgG in 0.01 M PBS. PI-I 7.4. containing 0.1% BGG was serially diluted with NRS IgG in 0.01 M PBS, PH 1.4. containing 0.1% BGG.
244
The maximum excitation and emission wavelengths in 0.01 M PBS buffer, pH 7.4, were 486 nm and 515 nm, respectively. The methanol solution of this product was stable at -20°C for at least 10 months of storage. Antibody dilution study A typical antibody dilution curve was shown in Fig. 1. Based on this result, the purified anti-cortisol IgG in 0.01 M PBS, pH 7.4, containing 0.1% BGG solution was considered most suitable for fluorescence polarization immunoassay at a final dilution of 1 : 600. Incubation time and replacement study with inert cortisol Fig. 2 shows the time-course changes of the polarization during the incubation and replacement study with inert cortisol. Experimental methods were similar to those described in the section antibody dilution study. To replace with inert cortisol, 100 ng of cortisol were added to the assay tubes. Significant
60 Incubation Fig.
2.
Tnne-course
replacement assay 1
tube.
1920;
study Final A -------A,
of
changes
with
inert
antibody 1
: 3840;
time
in polarization cnrtisol.
dilution: --
To
(mln)
during replace
l-,
1
, NRS
IgG
mcubatmn
with
: 240;
mat ~
as a control.
at various cortisol,
.
1
100
: 480:
antibody
concentrations
ng cortisol =--
n. 1
was
added
: 960;
-__
and to
the ,,
245
decrease of the polarization was observed at a final antibody dilution 1 : 480 and 1 : 960. From these results, a suitable incubation time was decided as 60 min and final dilution of antibody was selected at 1 : 600. Specificity
Table I lists the cross-reaction 50% binding. Calibration
curve for the present
of the antibody
used with other
steroids
at
immunoassay
As illustrated in Fig. 3, the calibration curve for the fluorescence polarization immunoassay was obtained by plotting the polarization with arbitrary units (polarization value B - polarization value A) versus the logarithm of inert cortisol concentration. The minimal amount of cortisol detected was 1.5 ng/ tube and the measurable range was from 1.5 to 100 pg/dl. Precision
Intra- and inter-assay variations were determined from assays of serum samples with low and high cortisol concentrations. The intra-assay coefficient of variation (C.V.) for the analysis of 6 samples from high (mean 18.5 pg/dl)
18%
160
1.56
3.13 cortisol
12..5
25.0
concentration
6.25
(ug/dl)
50.0
100
Fig. 3. Calibration curve for the current immunoassay of cortisol obtained by plotting the polarization with arbitrary units (polarization value B -polarization value A) ver‘sus the logarithm of inert cortisol concentration.
246 TABLE
I
CROSSREACTION THE PROPOSED Percentage
OF OTHER METHOD
crossreaction
STEROIDS
was calculated
WITH PURIFIED
displacing
50% of labeled
compound
mass of steroid
displacing
50%
compound
tested
Cortisol Progesterone Corticosterone Deoxycortisol DOC
IgG MEASURED
BY
as follows
mass of cortisol
Compound
ANTI-CORTISOL
of labeled
x 100
% crossreaction 100 13.3 9.3 8.1