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

Fluorescence polarization immunoassay for cortisol.

241 Clinica Chimica Acta, @ Elsevier/North-Holland 92 (1979) 241-247 Biomedical Press CCA 9986 FLUORESCENCE POLARIZATION YOSHIHARU KOBAYASHI...
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