109

CIinica Chimica AC& 86 (1978) 109-120 0 ElsevieriNorth-Holland Biomedical Press

CCA 9410

A SIMPLE RADIOIMMUNOASSAY

J. SETH and LYNDESAY

FOR PLASMA CORTISOL

M. BROWN

~epartrne~lt of Clinicaf Che~~~t~y, The Royal Infir~ffry, ~di~~urg~, EH3 9YW (U.K.) (Received December 19t.h, 1977)

Summary A simple radioimmunoassay (RIA) for plasma cortisol is described which combines the advantages of (i) direct analysis of untreated plasma samples, (ii) use of solid-coupled anti-cortisol antibodies and (iii) use of a gamma-labelled radioligand. The reagents are relatively easily prepared and stable, and the analysis can be completed in 4 h. Inter-assay precision (C.V.) is 8-il%. Critical ex~ination of specificity using high pressure liquid chromato~aphy showed that 23-35s of the immunoassayable material in plasma was not cortisol. RIA results on samples collected under basal conditions were on average 40 nmol/l lower than fluorimetric results, while in insulin hypoglycaemia and synacthen (ACTH) stimulation tests, this difference increased to over 100 nmol/l. The RIA is technically more simple than fluorimetric, competitive-proteinbinding, and many RIA methods, and can be used with advantage in the routine investigation of adrenocortical function. However, using the present antiserum, the RIA is not applicable to investigations on patients receiving metyrapone, nor in suspected cases of congenital adrenal hyperplasia.

Introduction In many clinical chemistry laboratories plasma cortisol is measured by fluorimetry [ 11. Since the early 1970’s however, competitiverprotein binding (CPB) and radioimmunoassay (RIA) methods have been increasingly employed, the advantages claimed for these methods being increased sensitivity and specificity. These advantages are in part offset by the increased technical complexity of many CPB and RIA methods. Many require some form of sample pre-treatment, e.g. solvent extraction [2,3], protein precipitation [4] or heat denaturation [ 5,6,7] prior to analysis. Separation of free and antibody-bound hormone in many RIA methods involves the use of dextran-coated charcoal, which requires careful timing of the separation step [2,4]. Many CPB and RIA methods

110

require the use of a 3H-radioligand and liquid scintillation counting, which in comparison with gamma counting is expensive in time and materials. A simplified cortisol radioimmunoassay which overcomes many of these drawbacks has been described by Rolleri et al. [S], while Read et al. [9] have described the use of a radio-iodinated ligand to simplify radioactivity counting. This report describes a further simplification of cortisol RIA. The laboratory evaluation of the method and preliminary experience in its clinical application are described. Materials

and methods

Anti-cortisol serum : Rabbit anti-cortisol-3-(0-carboxymethyl)-oximino-BSA serum, bleed RlB3, was obtained as a gift from Dr. G. Read, The Tenc:us Institute, Cardiff. Full details of the preparation and properties of this antiserum have been described elsewhere [4]. Whole anti-cortisol serum was covalently coupled to cyanogen-bromide activated cellulose by the method of Wide [lo] slightly modified so that the coupling was performed in sodium borate buffer, pH 8.6, 0.1 mol/l. The solid-coupled antibodies were stored in assay buffer at a 1 : 100 dilution (expressed as a dilution of the whole antiserum), and were stable for at least 6 months at 4°C. Charcoal-extracted serum was prepared by the procedure of Mitsuma et al. [ 111, with the addition of a final filtration through a 0.4+m (average pore size) filter to remove charcoal fines. This procedure removed 99.6% of endogenous cortisol, as indicated by the recovery of 3H-cortisol added to the serum prior to extraction. Cortisol standard solutions: Working standards covering the concentration range 0 to 2000 nmol/l were prepared in charcoal extracted serum and were stored in aliquots at -20°C. Under these conditions, the standards were stable for at least 2 months. Radiolabelled steroids: Cortisol-3-(0-carboxymethyl)oximino-[ “‘Iliodohistamine, (Cortisol-3-‘251), was prepared by the procedure of Hunter et al. [12] modified as described by Read et al. [13] to improve the yield and purity of the product. The thin layer chromatographic purification of the product gave a single peak of radioactivity, Rf approximately 0.4 while non-iodinated cortisol3-(0-carboxymethyl)oximino-histamine had an Rf of approximately 0.2. The recovery of “‘1 activity in the product (theoretical specific activity 1.8 Ci/ pmol) was 40% and the product was stable for at least three months when stored in ethanol at 4°C. Cortisol-3-carboxymethyloxime for use in preparing cortisol-3-1251 was prepared by the procedure of Janoski et al. [ 141, which gave a product of melting point 215”C, yield 23%. [ 1,2,6,7 ,-3H(N)] Cortisol, specific activity 91 Ci/mmol was obtained from the Boston, Mass. 02118, and was pure as New England Nuclear Corporation, judged by high-pressure liquid chromatography. Assay diluent: Sodium phosphate/citrate buffer, pH 4.0, 0.1 mol/l [15] containing 0.1% gelatine and 0.02% thiomersal as preservative was used as diluent. Assay tubes: All incubations were performed in glass or plastic round bottomed tubes, 77 mm X 25 mm O.D.

111

- CORTISOL

CORTISOL

0 Fig.

1.

Standard

tisol

as tracers.

Procedure

ADDED

using

anti-cortisol-3-BSA

for plasma cortisol

tube)

10.0

1.0

0.1 cumes

(],nwl

serum

(RIB3)

100 with

cortisol-3-1251

and

[1.2,6,7-3H]cor-

radioimmunoassay

All incubates were prepared in duplicate, samples from adrenocortical stimulation tests being analysed undiluted and at a 1 : 4 dilution in charcoalextracted serum. Solid-coupled antibody at a dilution of 1 : 4000 in diluent (200 ~1) was dispensed into assay tubes from a stirred suspension. Cortisol standards in charcoal-extracted serum (20 ~1) or serum/plasma samples (20 ~1) were added to the appropriate tubes, followed by cortisol-3-‘*‘I (20 000 cpm in 100 ,LI~diluent). After agitation on a vortex mixer the incubates were either stood overnight at room temperature, or mixed continuously on a multi-vortex mixer for 3 h at room temperature. Incubation was terminated by the addition from a repeating syringe dispenser of 2.0 ml sodium chloride solution (0.1 mol/l) containing 0.3% Brij (Techicon Corporation). The tubes were centrifuged for 15 min at 1500 X g, and the supernatants discarded by inverting the tubes smartly. Without turning the tubes upright, the tube tops were blotted dry on an absorbent pad, and the tubes taken for gamma counting. Sample results were interpolated directly from the standard curve (Fig. 1, ‘251-cortisol curve). Results Selection

of assay conditions

(a) Buffer

type and pH

In order to permit analysis of plasma samples introduced directly into the RIA incubate, the assay diluent was selected to reduce to a minimum the binding of cortisol to plasma proteins, without inhibiting the antigen-antibody reaction. Fig. 2 shows the effect of pH in sodium citrate/phosphate buffer, 0.1 mol/l, on the binding of cortisol to plasma proteins. Pregnancy plasma heat treated to inactivate corticosteroid binding globulin [ 51 showed a small but significant binding of cortisol at all pH values. In contrast, untreated pregnancy

112

2.0

3.0

4.0 IiEACTlON

5.0

6.0

7.0

l,tl

Fig. 2. Effect of pH on binding of cortisol to plasma proteins in sodium citrate/phosphate buffer (0.1 mol/I). Plasma concentration 6.6% 1, pregnancy plasma; 2, heat denatured pregnancy plasma; 3, no plasma added. Protein binding measured as % of added [3Hlcortisol in supernatant after addition of Sepbadex G-25 (F), 400 mg. Total reaction volume 2.9 ml.

plasma bound an increasing proportion of cortisol as the pH increased beyond 4.0. At pH values of 4.0 or less, binding did not differ between heat-treated and untreated pregnancy plasma. The effect of this residual cortisol binding, seen with both heat-denatured and untreated plasma at pH 4.0 or less, was small, and in the final assay resulted in a reduction of 1 to 3% in antibody binding at all points on the standard curve when standards in buffer were replaced by standards in charcoal-extracted serum. In order to compensate for this residual binding, assays were performed using standards in charcoal extracted serum to equalise protein concentrations in all tubes. Table I shows the effect of pH on the binding of cortisol-3-“‘1 to different solid-coupled cortisol antibodies. In general antibody binding decreased rapidly as the pH decreased below 4.0, although the rabbit antiserum RlB3 used in this study retained greater affinity for antigen at low pH values than did rabbit antiserum R5B5, or sheep antiserum FR31-1B. Sodium citrate/phosphate buffer, 0.1 mol/l, pH 4.0 was therefore selected as diluent for the reasons that it reduced binding by the plasma proteins to a minimum, while not seriously inhibiting binding by different anti-cortisol antibodies. Confirmation that interference from the plasma proteins was satisfactorily controlled was obtained by comparing cortisol concentrations in untreated plasma samples with concentrations in protein-free extracts of the same samples prepared by gel-filtration. Plasma aliquots (200 ~1) were chromatographed on columns (50 X 10 mm diameter) of Sephadex G-25 (fine) in assay diluent, after addition of [3H]cortisol to correct for chromato~aphic losses. Cortisol-binding proteins were eluted in the column void volume, well separated from cortisol. Aliquots of the

113 TABLE

I

EFFECT 3-BSA

OF

pH

ON

BINDING

ANTIBODIES

RlB3.

R5B5:

percentage

rabbit

of that

OF

IN SODIUM antisera.

at pH

1251

CORTISOG3-

TO

CITRATE/PHOSPHATE

FR31-1B;

sheep

CELLULOSE BUFFER.

antiserum.

For

COUPLED 0.1

each

ANTI-CORTISOG

mol/l

antiserum,

binding

is expressed

as a

7.0 ._.. I_ “..__

PH

5.5

100.0

100.0

100.0

4.5

100.7

98.0

94.0

4.0

96.7

91.6

91.0

3.0

92.3

51.6

54.2

2.3

73.6

24.5

32.5

_. --

pooled cortisol-containing fractions were taken for RIA using standards in diluent, the results being corrected for the recovery of [ 3H]cortiso1. Fig. 3 compares cortisol concentrations in both normal and pregnancy plasmas determined with and without gel-filtration. The calculated regression line does not differ significantly from the 45” regression line, and fails to demonstrate any bias in the direct assay due to the presence of plasma proteins. (b) ~ffdioZabe~led cortisol The final antibody dilution of 1 : 6000 for maximum response 3-‘251 (20 000 cpm, approx. lo-’ pmol/tube) was approximately

using cortisolten times that

800

600

0

400

200 CORTISOL

Fig.

3.

Comparison

chromatography patients;

0.

analysis)

=

(hatched).

of to

plasma 1.03

(gel

cortisol

remove from

600

(nmol.

1)BY

800 GEL

concentrations

plasma

proteins.

non-pregnant

filtration

analysis)

in plasma For

females -

1000

1,200

FILTfWTION

8.8,

samples

experimental or

does

from not

males. differ

measured details, The

see

with

and

text.

calculated

significantly

from

without

o, plasma regression the

45O

gel filtration from line,

pregnant (Direct

regression

line

using [3H]cortiso1 (20 000 cpm, 0.3 pmoljtube). Standard curves using 3H- and ‘251-labelled radioligands are compared in Fig. 1. The response of the “% system to a given mass of added cortisol was approximately one tenth that. of the “H-system, ~though still more t,han adequate when the sample volume was correspondingly increased. Varying the mass of cortisol-3-lZ51 per tube over the range 0.5 to 4.0 times that normally used had no significant effect on the characteristics of the standard curve. Counting times with the 12sI-system were one minute or less, depending on the age of the label and counting efficiency. In contrast, counting times with the 3H-system were 2 min per tube, to which must be added the additional time and labour of preparing aliquots of the supernatants for liquid scintillation counting. (c) Reaction time Antibody binding of cortisol-3- “5I reached a maximum after 2 h incubation at room temperature with continuous mixing. Without mixing, maximum binding was reached in 4 h, although it was generally more convenient to perform such incubations overnight. Standard curves prepared by mixing for 3 h, or by standing overnight did not differ significantly. Assessment

of assay performance

(d) Reeouery

The recovery of cortisol added to 17 different sera or plasma specimens to give concentrations in the range 250-2000 nmol/l was 100.7 i- 3.2% (mean ? S.E.M.). Recovery similarly calculated for 4 plasma specimens from t,hird trimester pregnancies was 98.9 -f-2.7% (mean i S.E.M.). (e) Precision Data illustrating intra- and inter-assay precision are shown in Table II. The mean values for pools A and C demonstrate in addition satisfactory long-term linearity and recovery. ff) ~peeific~ty Table III summarises relative reactivities of several steroids in the assay. Prednisolone and 11-deoxycortisol showed extensive cross reactions, with 17cyhydroxy progesterone and cortisone showing lesser but still appreciable cross reactions. Specificity was further investigated using high-pressure liquid chromatography (HPLC) to separate cortisol from potentially interfering compounds. Dichloromethane extracts of plasma samples containing [ 3H] cortisol were chromatographed under the conditions summarised in Fig. 4. Eluate fractions (0.7 ml) were evaporated to dryness, taken up in chinos-extracted serum, and assayed for cortisol and 3H activity. Specificity was assessed by examination of the chromatograms and by comparison of the specific activities (“H cpmjmass) of cortisol in the unextracted plasma, in the dichloromethane extract, and in the pooled eluate fractions containing cortisol. Fig. 4 shows the chromatograms of a plasma specimen from a pregnant patient, and is typical of the ~hromato~~s of plasma extra&s from non-

115 TABLE

II

PRECISION

OF

Intra-assay of

PLASMA

precision

CORTISOL

(S.D.)

RADIOIMMUNOASSAY

is calculated

as JXd2

f2N

where

d is difference

between

duplicates

in i? Pairs

determinations. .._

1ntra-assay

Concentration 190

_~

~~.

s

range

550

550-l

._ 877

325

S.D.

32.2 (%)

No.

58.5

9.8

of duplicates

6.6

45

Inter-assay

55

Plasma Pool

Mean

eortisol

(nmolfi)

A *

Pool

No.

10.7

of determinations

* Pool

pool

with

4 volumes

**

66.2 8.4 15

14

B diluted

c

790 8.4

15

A is 6 volumes

Pool

37.8

29.8 (90)

B

450

279

S.D. C.V.

550

_

Mean C.V.

_~-

(nmol/l)

of charcoal-extracted

serum

(theoretical

values

270

~IllOl/l). **

Pool

C is pool

B with

cortisol

(345

nmolfl)

added

(theoretical

value

795

nmol/l).

pregnant patients. A small peak corresponding to 3 to 5% of the applied mass was invariably seen at an elution volume of one half that of cortisol, corresponding in chromatographic mobility to cortisone. No mass peaks were detected at mobilities corresponding to other cross-reacting steroids. Table IV compares specific activities of cortisol in unextracted plasma, and in dichloromethane extracts of plasma before and after HPLC. The relatively low specific activities of cortisol in the dichloromethane extract compared with the fractions obtained after HPLC indicate the presence of interfering material TABLE

III

RELATIVE 3-l

251

REACTIVITIES

Relative binding

OF

STEROIDS

IN SOLID-COUPLED

ANTICORTISOL-3-BSA/CORTISOG

SYSTEM reactivities by

15%

are calculated

from

that

in the

as the relative zero

standard.

molar (p),

quantities (np)

denote

of each

steroid

parallel

and

required

to reduce

non-parallel

curves

tracer respec-

tively .

._ Steroid

Relative

Cortisol

1.00

11-Deoxycortisol

0.61

(P)

Prednisolone

0.44

(P)

17a-Hydroxyprogesterone

0.21

(np)

Cortisone

0.15

(P)

Prednisone

0.07

(P)

Corticosterone

0.016

Dexamethasone

0.006 _.._______-_.

The

following

pregnanetriol. aIl010lle.

steroids

.-.__ had

pregnanetetrol,

relative

_---

(P) ~___

..____~ reactivities

aldosterone.

~__

reactivity

of

0.003

progesterone.

_.

_ ___.____~__

or less:

cortol,

._~__. cortolone.

11-oxo-androsterone,

.-...-_ tetrahydrocortisone,

androsterone,

aetiochol-

116

0 0

2

x

6

*

10

F’r:ici,z>n

Fig.

4.

~hrom~togr~Ins

(0.2

MCi,

flow

rate

2.4 1.4

pmol

of per

ml/min,

ml).

0.7-ml

12

14

1s

16

22

20

Nsr. lc1.7 n:li methylene

1~1

dichloride

extract

fractions

extract

injected.

of

equivalent

pregnancy to

200

plasma

containing

~1 plasma.

Inlet

(3fI]cortisol

pressure

10.0

bar,

collected.

in the extract accounting for 9-22% of the mass chromatographed. This extent of interference is not accounted for by the small peak referred to above, containing 3--5% of the mass chromato~aphed. Furthermore, the low specific activities of cortisol in unextracted plasma compared with the whole dichloromethane extracts indicate the presence of additional interfering material in unextracted plasma. Assuming 100% purity for the cortisol fractions after HPLC, 23--35% of the material assayed in unextracted plasma was not cortisol. Additional information on assay specificity was obtained from paired comparisons with the results of fluorimetric analysis [ I] . Table V shows that for specimens collected under basal conditions, RIA results were on average 40 nmol/l lower than fluorimetric results, but that for specimens collected under conditions of adrenocortical stimulation, this difference increased to 100-150 nmol/l. Patients on dexamethasone showed adrenocortical suppression judged by both fluorimetric and RIA results, as did patients on prednisolone, despite the higher cross reactivity of this drug. RIA and ~uorimetric results in two patients on prednisolone were 118 and 128 nmol/l respectively

TABLE

IV

SPECIFICITY OF

pmolf (ii)

A

OF

PLASMA

[3H1CORTISOL

DICHLOROMETHANE

CORTISOL IN

RIA,

PLASMA EXTRACT

ESTlMAT~~

MEASURED OF

BY

PLASMA

AND

FROM RIA (iii)

OF

SPECIFIC (i)

ACTIVITY

NON-EXTRACTED

CORTISOL

IN ELU.4TE

$H

cpm/

PLASMA, FROM

HPLC

COLUMN For

experimental

details

see text

and

Fig.

4. ..-

Plasma

Cortisol

Specific

specimen

(nmol/lf

_

activity,

f3H

cpn~/pmol) _-

Nun-extracted

DichIorom~th~me

Cortisol

plasma

extract

elLlate

A

414

199

239

306

B

745

173

205

225

C

690

372

432

511

D

440

747

986

1196

in HPLC

117 TABLE

V

PAIRED ETRY

COMPARISONS AND

RIA

OF

UNDER

ADRENOCORTICAL

PLASMA

BASAL

FUNCTION

Specimen

Range

collection

observed

time

(RIA)

CORTISOL CONDITIONS

LEVELS AND

(nmol/l)

IN

SOME

MEASURED

BY

FLUORIM-

IIYPOTHALAMIC-PITIJITARY-

TESTS Difference

between

results

(Fluorimetry-RIA)

MeaIl

S.E.M.

difference

difference

of

Observations

__. Specimens

collected

0990

h

2200

h

Specimens

under

basal

conditions

121-966 25-497 from

patients

40

18

19

39

16

18

on dexamethasone 44

7

8

160-420

42

15

9

+30

187-640

95

27

8

+60

190-750

132

36

8

500-800

149

27

9

660-890

154

22

8

14.-41 Insulin-hyoglycaemia -5

tests

*

+90 +120 Synacthen -5

stimulation **

tests 196-662

72

7

+30

232-828

123

52

46

7

+45

248-952

103

45

7

. .._._ ” . . * **

Minutes

after

insulin

Minutes

after

synacthen

injection

(0.15

injection

p/kg

(250

i.v.).

pg i.m.).

(dose 80 mg/day) and 165 and 136 nmol/l (dose 10 mg/day). The greater specificity of the RIA was particularly evident in investigations on a patient receiving spironolactone. Fluorimetric results were greater than 1600 nmol/l, with apparent lack of overnight suppression on dexamethasone (2 mg). RIA of the same samples showed a normal diurnal variation of 200 to 400 nmol/l, with suppression on dexamethasone. Discussion The principal features contributing to the simplicity and reliability of this assay are (i) the use of diluent of low pH to permit the direct analysis of an unextracted plasma sample, (ii) the use of cellulose-coupled antibodies to simplify the separation of free and antibody-bound hormone and (iii) the use of 1251labelled cortisol to simplify radioactivity counting. Use of sodium citrate buffer of low pH in the direct analysis of plasma cortisol was first described by Rolleri et al. [S], who used cellulose-coupled anticortisol-21-BSA antibodies and a 3H-cortisol radioligand. Our experience demonstrates that a low pH diluent is also suitable for the solid-coupled anticortisol-3-BSA antibodies/cortisol-3-‘251 system, even though a larger volume of the plasma sample is required. The small residual binding shown by both untreated plasma and heat-trea~d plasma under the conditions of our assay may be due to albumin [ 161 but is effectively compensated for by use of standards in charcoal-extracted serum. No significant interference from plasma proteins

118

could be detected in samples from third trimester pregnancies, where raised corticosteroid-binding globulin levels might be expected to cause most serious interference. Incubation at low pH is much more convenient in practise than commonly used alternatives for eliminating plasma protein interference, such as solvent extraction [2,3], ethanol precipitation [4], or heat denaturation [5,6,7]. Furthermore, results reported here and elsewhere [8] demonstrating the suitability of a low pH for assays using different anti-cortisol sera raised against different immunogens in different species, and using both 3H and radio-iodinated ligands, suggest that this might be a generally applicable approach. Use of solid-coupled antibodies adds greatly to the convenience of the assay in that the separating system and antibody are combined in a single reagent. Free and antibody-bound hormone can be separated with minimum disruption of the antigen-antibody complex, as shown by the small decrease in antibody binding (0.6%) for a delay of 30 min between adding wash solution, and centrifuging and decanting. In contrast, disruption of the antigen-antibody complex using dextran-coated charcoal separation necessitates careful timing of the separation step [ 21. Use of a radio-iodinated ligand provides important advantages in addition to the savings in time and materials in radioactivity counting. The increased antibody titre with cortisol-3- i2’I decreased antibody consumption, while the tenfold decrease in sensitivity permitted the use of larger volumes of sera, with a consequent increase in sampling precision. The minimum detection limit using the ‘2sI-system, (20 nmol/l) was still more than adequate for clinical use. Cross-reactivities with other steroids are generally greater with ‘2sI-ligands than with 3H-ligands [ 171, and the homologous anti-cortisol-3-BSA/cortisol-3“‘1 system is no exception to this [9,18]. Although appreciable cross reactions were observed with other steroids in the present assay, only cortisone would be identified on the basis of HPLC as causing measurable interference in samples of normal plasma. Of greater concern was the evidence that on average, 30% of the material in extracted plasma was not cortisol. The compound tentatively identified as cortisone would account for only a small part of this interference. Polar metabolites of cortisol which are not extracted by dichloromethane might account for an additional part, and preliminary experiments have shown that solvolysable material in plasma, possibly sulphates of cortisol or its metabolites, account for a substantial part of the interference in some plasmas. The relatively small difference between the results of this RIA and fluorimetry for samples collected under basal conditions is similar to the experience reported with other radioimmunoassays [2,6,7,8]. As on average, 41% of the material assayed by the Mattingly procedure is not cortisol [19], the small difference between methods described here is consistent with the evidence indicating that in the current procedure, 30% of the immunoassayable material in plasma is not cortisol. Specimens collected during insulin hypoglycaemia tests, and during synacthen (ACTH) stimulation tests gave much lower results by RIA than fluorimetry. This probably reflects adrenocortical secretion of steroids other than cortisol which are detected by fluorimetry. Corticosterone, for example, is not detected by RIA, but gives approximately twice the fluores-

119

cence of cortisol per unit mass under the conditions of the ~attingly procedure. A similar exaggeration of the difference between RIA and fluorimetric results under conditions of adrenal stimulation has been reported by Carr et al. [‘7] and Kumar et al. [ 201, and indicates the need for redefinition of normal responses in stimulation tests using more specific RIA measurements as an index of response. Although this RIA is in general more specific than fluorimetry, the specificity characteristics of the present antiserum make it unsuitable for investigations on patients receiving metyrapone, or on patients in whom congenital adrenal hyperplasia is suspected. These are also circumstances in which simple CPB and fluorimetri~ methods can give misleading results due to interference from other compounds. It is probable that these particular sho~comings in RIA specificity could be overcome by careful selection of antiserum, and test bleeds of sheep anti-co&sol sera have shown a lower cross reactivity with other steroids in our system. The cross reactivity with prednisolone in this RIA did not appear to be a serious problem, as the two prednisolone treated patients described here showed adrenal suppression by both RIA and fluorimetric measurements. The present RIA is more convenient than fluorimetry, especially for the analysis of large numbers of samples, and is also more simple than CPB methods and ot,her non-extraction methods for cortisol RIA [6,7,8]. The stability of the reagents and their relatively low cost makes this method an attractive alternative to simple RIA methods using kit reagents [ 211. Sensitivity and precision are comparable with, and in some cases better than is achieved with more complex methods, and although specificity is not complete, this limitation has not proved a disadvantage in practice. Acknowledgements We are most grateful to the following for their contributions to this work: Dr. G. Read, of the Tenovus Institute Cardiff, for supplying antiserum RlB3, Dr. Brian Morris, of the University of Surrey, for supplying test bleeds of sheep anti-cortisol serum, and Dr. W.M. Hunter, of the MRC Radioimmunoassay Team, Edinburgh, for provision of facilities for the preparation of ~diolabelled cortisol. Part of the work described in this paper will form part of the thesis to be presented by L.M. Brown for the award of Fellowship of the Institute of Medical Laboratory Sciences. References 1

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2

Ruder,

3

Dash,

4

Fahmy,

D. (1962)

H.J.. R.J.,

5

Murphy,

6

Foster,

7

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8

Rolleri,

9

Read,

GUY, England,

D..

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B.E.P. L.B.

P.J.,

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Millax,

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R.P.

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Zannino,

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Fahmy,

(1972)

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and

Hillier,

S.G.

R.T. and

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41,

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Dunn,

15. M.B.

and

(1975)

Path& Lips&t.

Ann,

G.D.

(1975)

35,

219-224

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26,647-661

26,267-280

365-368 Clin. R.

Bioehem.

(1976) Clin.

Clin.

Bioehem.

14,207-211 Chim.

Acta

14,343-349

66,

319-330

120 10 il 12

13 14 15 16 17 18 19 20 21

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