Solid phase radioimmunoassay of plasma aldosterone

809

SOLID PHASE RADIOIMMUNOASSAY OF PLASMA ALDOSTERONE

I

Charles A. BIZOLLON, Jean-FranGois RIVIERE Andr~ FAURE and Bruno CLAUSTRAT.

, Paul FRANCHIMONT

2

,

Laboratoire des Radio~l~ments (D~partement de Radioimmunologie) Pharmacie Centrale des Hospices Civils de LYON, 69425 ~YON CEDEX 3 (FRANCE). Laboratoire Central de Biochimie (Pr. R. MALLEIN) D~partement d'Hormonologie, HSpital de l'Antiquaille, 69321 LYON CEDEX 1 (FRANCE). 2 Laboratoire de Radioimmunologie, Institut de M~decine, Universit~ de Liege (BELGIQUE).

Received:

2/18/74

ABSTRACT A solid phase radioimmunoassay for the measurement of aldosterone in plasma is described. The antiserum was produced by immunizing rabbits with 3-carboxymethyloxime of aldosterone-18-21-diacetate coupled to bovine serum albumin. This antiserum was covalently linked to an iminocellulose according to the procedure of Wide and used in the assay at a 1/1050 final dilution. It contained antibodies with association-constant of I.I x I0 IO M -I and was fairly specific since with the exception of aldosterone acetates, none of the tested steroids cross-reacted more than 0.05 per cent. Aldosterone was extracted with dichloromethane, purified by paper chromatography, then submitted to the assay. The intra-assay reproducibility varied between 4 and 13 % and the inter-assay reproducibility between 13 and 2 1 % . The least detectable amount was 5 Dg per tube. This method is very simple and, with the exception of the chromatographic step, can be completed in half a working day. INTRODUCTION Numerous radioimmunologic methods have been proposed for the assay of plasma aldosterone°

All of them require the classical processes

of separation of free and bound fractions

Volume 23, Number 6

: adsorption or extraction of

S T E R 0 1D S

June, 1974

81o

ST E RO I DS

the free fraction by means of Florisil (3,4,5,6),

scintillation mixture

(1,2), dextran-coated

charcoal

(7) or salting out of the antibody-

bound fraction with ammonium sulfate

(8,9,10).

In setting up the aldosterone radiolmmunoassay,

we found that

part of the blank could vary according to the separation method employed (dextran-coated

charcoal or ammonium sulfate). We, therefore,

decided not to add any supplementary reagent to the incubation medium and consequently adopted a solid-phase

system.

Such systems were already applied to the radioirmnunoassay of various steroids

: Abraham et al. (11,12) used antibody-coated

tubes according to the method of Cart and Tregear (14) recommended

(13). Mikhail et ~i.

the use of antibodies polymerized with ethylchloro-

formate by the method of Avrameas covalent-binding

plastic-

of antibodies

(15). Moore and Axelrod

(16) proposed

to Enzacryl AA. Loriaux et al.

used antibodies trapped in polyacrylamide-gel.

Finally,

(17)

Bodley et al.

(18) reported the use of antibodies covalently linked to arylamine glass particles, iminocellulose,

We adopted covalent-binding as suggested by Axen et al.

to the radioimmunoassay et al. (21) and Hendrick

of the antibody to an (19) and earlier applied

of protein hormones by Wide (20), Franchimont (22). MATERIALS

Ethyl acetate, acetone, dichloromethane (analytical grade) and benzene, ethanol, methanol (spectro grade) (Merck) were used without further purification ; pyridine (Merck) was freshly distilled ; carboxymethoxylamine hemihydrochloride (Aldrich Chemical Co., Inc.), N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (K. and K. Laboratories, Inc.), bovine serum albumin (BSA), fraction V (Armour Pharmaceutical Co), cellulose microcrystalline for thin layer

ST E R O I D S

811

chromatography (Merck), cyanogen bromide (Prolabo), Tween 20 (Touzart et Matignon), Freund's complete adjuvant (Difco Laboratories) and Instagel (Packard) were used as supplied. d-Aldosterone was kindly supplied by Ciba Laboratories (Paris). Other non-radioactive steroids were obtained from Ikapharm. 1,2-3H-aldosterone-~8,21-diacetate was obtained through a 24 hour acetylation of 1,2- ~H-aldosterone 35 Ci/mM (Rad.iochemical Centre) at 37°C in pyridine/acetic anhydride 2/I and chromatographic purification on Whatman N ° I paper in the system cyclohexane/benzene/methanol/ water : 5/2/5/I. Cellulose was activated by stirring 15 g of cellulose in 500 ml of a saturated CNBr solution whose pH was maintained at I0.5 ! 0.5 through a careful addition of 1 N NaOH. After 5 minutes, the solution was quickly filtered through a B~chner funnel. The activated cellulose was then washed with cold water, dried with acetone and stored at -20°C. The Bush B5 system (benzene/methanol/water : 10/5/5) was freshly prepared with solvents from newly opened cans. Whatman N°40 paper was washed for 24 hours with the mobile phase of the B5 system, dried at room temperature and used immediately. 0 . O 1 M phosphate buffer, pH 7.4, contained 9 g of NaCI, 5 g of BSA and 5 ml of Tween 20 per liter. All the glassware was immersed in a Pyroneg solution (Diverscy) for 24 to 48 hours, rinsed with water, then acid washed, rinsed again with glass-distilled water and finally dried. Evapomix (Buchler Instruments) with a plastic holder allowed simultaneous shaking of 80 tubes. For the radioimmunoassay, all the reagents were delivered through Eppendorf pipettes. METHOD S Preparation of the antibody The method of Bayard et al. (I) was applied with slight modifications. The carboxymethyloxime of aldosterone-diacetate was prepared with tritium-labelled aldosterone-diacetate (500.000 dpm/O.l mmole) and coupled to BSA (80 mg) during a 2 hour shaking after dissolution in 1 ml of pyridine/distilled water I/I and addition of 200 mg of EDC. The resulting mixture was dialysed against running water for 12 hours, homogenized and diluted to lO ml with distilled water. Counting of an aliquot gave a molar ratio aldosterone/ BSA of 5. The antigen was then divided into 0.5 ml fractions and stored at -20°C, so that the whole immunization be achieved with the same antigenic material. ~ For each injection, a fraction was thawed, diluted with 1.5 ml of saline and emulsified in 4 ml of Freund's adjuvant. Six rabbits were immunized with multiple site injections (subcutaneous and intradermal) of 1 ml of the emulsion. The animals were injected fortnightly during 2 months, then monthly during 3 months and then every 4 months. They were bled lO to 12 days after each injection. Two rabbits survived

812

ST E R O I DS

and produced antibodies with sufficient titer to be used in the assay, the first animal 4 months after the first injection, and the second, eighteen months later. Preparation of the immunosorbent Wide's method (20) was used. After immunoadsorption for 48 h. at 4°C with I00 mg of BSA per ml of serum the precipitate was discarded ; globulins were salted out with 18 % NapSO 4 (W/V) and dissolved in 5 ml of 0. I M NaHCO 3. Three ml of 0. I M NaRCO 3 and 750 mg of activated cellulose were added to 2.5 ml of this solutlon. After shaking for 24 hours at room temperature, the immunosorbent was washed according to Wide (20), diluted in I0 ml of assay buffer and stored at -20°C in I ml fractions. At the time of the assay this stock suspension was properly diluted (I/37.5) and homogenized on magnetic stirrer. Assay procedure To two ml of plasma, containing 25 to 500 pg of al~ro--~], "9"O'Odpm of 1,2-3H-aldosterone (3 pg) were added. The sample was extracted by I0 volumes of dichloromethane. The organic phase was washed with 2x2 ml of bidistilled water and dried at 37°C under a stream of nitrogen. The residue was dissolved in 50 lUl of . methanol and transferred to a pre-washed Whatman N ° 40 paperl Simultaneously, a 2 ml distilled water-extract (blank) as well as a 40 /ug aldosterone spot were applied to the paper. After 5 h. of equilibratio~ at 25°C, the chromatogram was run 5 to 7 h. in the B5-system. The reference spots were visualized under U.V. (250 nm) light, and the corresponding areas were cut out and eluted with methanol. The eluates were dried and dissolved in 300 /ul of assay buffer by means of a 30 min. shaking at room temperature. A 100 /ul fraction was used for recovery and 2 other fractions (100 and 50 /ul) were used for the assay. Standardization was achieved in triplicate by means of I00 ul aliquots of buffer assay solutions containing O, 25, 50, I00, 200, ~OO and 800 pg of aldosterone. In~nunosorbent suspension (500 /ul) was then added to each tube, whereas 3 tubes for the total activit~ determination received 600 /ul of assay buffer. All the tubes were stoppered with plastic caps and shaken on an Evap~mix for 2 hours at 37°C. They were cooled to 4°C and 7700 dpm of 1,2- H-aldosterone (20-25 pg) in 100 /ul of assay buffer were added ; they were shaken again for 2 hours at 4°C, then centrifuged 5 min. at 3000 g. The supernatant was aliquoted (0.5 ml) into counting vials and counted for 20 min. after addition of IO ml of Instagel. The activities obtained were substracted from the total activity and the percentage of the counts bound to the antibody was plotted against the amount of aldosterone. The values of unknown samples were read off the standard curve. Release of aldosterone by the immunosorbent was studied at IO°C or 37vC in the following way : test tubes containing 0.7 ml of the immunosorbent suspension and (a) 20 pg of 1,2- H-aldosterone or (b) the same quantity of cold aldosterone were incubated with continuous

ST E R O I D S

813

shaking until equilibrium was reached. They were then centrifuged and the supernatant of tubes "a" was discarded and replaced at the zerotime by the supernatant of tubes "b". The tubes were shaken again on the Evapomix and the activity of the supernatants was then studied as a function of time. Non-chromatosraphic radioimmunoassays were achieved on crude dichlorometh~ane extracts of plasma w~h~ch were only washed with distilled water and dried. RESULTS A comparison to cellulose

between antibody-titers

is shown in figure

before and after coupling

I. Coupling

loss in the titer, but the dilution

to cellulose

produced

curve of the immunosorbent

a steeper

slope than that of the crude serum. Non immune rabbit

globulins

coupled

terone

to activated

cellulose

had no affinity

a

had

for aldos-

in our assay conditions. We made a parallel

the uptake

(fig.

aldosterone 35 minutes

kinetic

2) and of the release

by the immunosorbent. and further

antibody-bound

incubation

fraction.

longer,

(fig.

A comparfson

through a one-step method

in

alter the

the dissociation

complex was relatively

rate

high. At low

to be reached was much

rate of the complex was practically

was also made between

of

3) of tritiated-

did not significantly

the time needed for equilibrium

but the dissociation

temperatures

At 37°C equilibrium was reached

At this temperature,

of the aldosterone-immunosorbent temperature,

study at different

standard

zero.

curves obtained

and through a late addition

of the labelled

hormone with a second 2-hour

incubation

method resulted

slope in the initial part of the standard

curve

(~200

in a steeper

pg) whereas

at 4°C (fig. 4). The two steps

the one step method

resulted

in a better

814

STE

ROI

DS

standard curve between 200 and 800 pg. The specificit~ of the assay depends upon the use of a highly resolutive chromatographic stev and upon a very low antibody crossreactivity. The specificity of this antiserum improved with time of immunization, but its affinity-constant

calculated from Scatchard Dlots did

not vary significantly and was in the range of I0 IO M -I. The percentage cross-reaction of various steroids is shown in table I. Antibody specificity was further investigated by means of a non-chromatographic radioi~m~unoassay.

Comparison between results obtained with and without chro-

matography appears in figure 5 : non chromatography produced a 24 % average overestimation and all samples but 3 were overestimated. The recovery of 1,2-3H-aldosterone added to plasma was 62 + 13% (mean ~ SD, n = 91). Samples with recoveries below 40 % were systematically discarded. Blank and sensitivity

: the blank was negligible, provided

solvents were used in an appropriate way. Sensitivity was calculated from I0 standard curves according to Feldman and Rodbard

(23) ; it was

5 pg at the 95 % confidence level (2,26 SD). Accuracy and precision : accuracy and intra-assay precision were determined by adding different amounts of aldosterone to 2 ml aliquots of a charcoal-treated plasma pool. Results appear in table 2. The value obtained for the plasma without added aldosterone was not significantly different from zero. The mean recovery (corrected for recovery of radioactivity) varied between 94 and 106 % and the coefficient of variation between 4 and 13 %. The inter-assay precision was estimated by

STERO

1DS

815

determining the coefficient of variation of the aldosterone contents of 2 different pools of plasma measured on different days. The obtained values were 70 + 15 and 240 + 32 pg/ml (mean + SD, n - 9) ; the coefficients of variation were then 21 and 13 % respectively, x Normal values obtained through this method were in good agreement with those found in the literature and ranged from I0 to 40 pg/ml for supine subjects. They ranged from IIO to 180 pg/ml after these subjects had been standing for 3 hours. Clinical results will be published elsewhere. DISCUSSION The specificity of the antibody immproved in the course of immunization as the cross-reactivities of cortisol, cortisone, corticosterone and 17,21-dihydroxy-4-pregnene-3,20-dione

decreased with

time when the 3 antisera were tested at the same time. There is no need to underscore the advantages of solid-phase antibody utilisation : simplicity and better reproducibility than with classical separation-methods

since no supplementary reagent

disturbs the steroid-antibody equilibrium.

The coupling to cellulose

is easy to achieve and the irmmunosorbent obtained proved stable for more than 6 months when stored in a freezer. Coupling to iminocellulose, however, resulted in a 2 or 5 fold loss in the titer of the antibody. This loss could depend either upon the fixation of the immunoreactive sites of the antibodies to the iminocellulose or Accuracy and precision were checked using the 2-steps method within the usuable part of the standard-curve (~ 200 pg) ; accuracy and precision of the l-step method were not investigated.

816

S T E R O I D S

upon steric inhibition of the coupled antibodies

(22). On the other

hand, this loss in titer seemed to go along with an increase in sensitivity, as shown by the steeper slope of the dilution curve after the antibodies had been coupled to activated cellulose (figure ]). This could be attributed to the dissociation of the possible steroid-antibody complexes as the inm~unosorbent is acidwashed after the coupling reaction. The endogenous steroids bound to the rabbit's antibodies might then be stripped off and one can imagine that the antibody population which might have been saturated is that which presented the highest avidity for aldosterone. The affinity constants calculated from Scatchard plots were not, however, significantly different before and after coupling of the antibodies to iminocellulose, but the calculated values are not to be compared since binding by the immunosorbent causes a phase-transfer of aldosterone. The incubation temperature

(37°C) was chosen for convenience.

This temperature does not alter aldosterone and induces quick binding by the irmmunosorbent (fig. 2). At 4°C the aldosterone-immunosorbent complex seems quite stable as shown in fig. 3. This observation allows us to improve the assay sensitivity through a late addition of the labelled hormone and through a second 2-hour incubation at 40C (figure 4). As to the non-chromatographic radioimmunoassay,

despite a

good correlation between aldosterone concentrations obtained through dichloromethane extracts of plasma which had or had not

ST E R O I D S

been submitted to chromatography

817

(figure 5), the small number of

these comparative assays has not made it possible yet to draw a reliable conclusion.

However,

lipids in the crude plasma extracts

generated a turbid incubation medium which might have disturbed the aldosterone-immunosorbent

equilibrium. REFERENCES

I. Bayard F., Beitins I.Z., Kowarski A°, Migeon C.J. J. CLIN. ENDOCRINOL. 31:1 (1970).

:

2. Ito T., Woo J., Haning R., Horton R° : J. CLIN. ENDOCRINOL. 34:106 (1972). 3. Waldhaust W., Haydl H., Frishauf H. : STEROIDS 20:727

(1972).

4. Farmer R.W., Roup W.G. Jr., Pellizari E.D., Fabre L.F. Jr. J. CLIN. ENDOCRINOL. 34:18 (1972). 5. Farmer R.W., Brown D.H., Howards D°Y., Fabre L.F. J.~CLIN. ENDOCRINOL. 36:461 (1973). 6. Corvol P., Menard D., Bertagna X. : ANN. ENDOCR. 34:57 (1973).

:

:

(PARIS)

7. Ekins R.P., Newman G.B., Piyasena R., Banks P., Slater D.H. J. STEROID BIOCHEM. 3:289 (1972). 8. Mayes D., Furuyama S., Kem D.C., Nugent C.A. ENDOCRINOL. 30:682 (1970). 9. Martin B.T., Nugent C.A.

: J. CLIN.

: STEROIDS 21:169 (1973).

I0. Gomez-Sanchez C., Kern D.C., Kaplan N.M. 36:795 (1973).

: J. CLIN. ENDOCRINOL.

II. Tillson S.A., Thorneycroft I.H., Abraham G.E., Scaramuzzi R.J., Caldwell B.V., IMMUNOLOGIC METHODS IN STEROID DETERMINATION Editors F.G. Peron and B.V. Caldwell. Appleton-Century-Croft. New York. 1970, p. 127. 12. Abraham G°E., Grover P.K. PRINCIPLES OF COMPETITIVE PROTEINBINDING ASSAYS. Editors W.D. Odell and W.H. Daughaday. Lippincott C ° . Philadelphia and Toronto. 1971, p. 140.

818

STEROI

13. Cart K.J., Tregear G.W.

DS

: Science 158:1570 (1967).

14. Mikhail G., Wu Ch., Ferin M., Wande Wiele R.L. STEROIDS 15:;33 (1970). 15. Avrameas S., Ternynck T.

: J.

16. Moore P . H . ,

: STEROIDS 20:199

Axelrod

L.R.

:

BIOL. CHEM. 242:1651

17. Loriaux D.L., Guy R., Lipsett M.B. 36:788 (1973).

(1967).

(1972).

: J. CLIN. ENDOCRINOL.

18. Bodley F.H., Chapdelaine A., Flickinger G., Mikhail G., Yawerbaum S. and Roberts K.D. : STEROIDS 21:1 (1973). 19. Axen R., Porath J., Ernbach S. : NATURE 214:1302 (1967). 20. Wide L. : ACT. ENDOCR.

(Kbh.) Suppl.

(London)

142:207 (1969).

21o Franchimont P., Hendrick J.C., Demeyts P. RADIOIMMUNOASSAY METHODS Editors K.E° Kirkham and W.M. Hunter. Churchill Livingstone. Edinburgh and London. 1971, p. 416. 22. Hendrick J.C° : THESE DE DOCTORAT ES SCIENCES BIOMEDICALES EXPERIMENTALES. Universit~ de Liege (]972). 23. Feldman H. and Rodbard D. PRINCIPLES OF COMPETITIVE PROTEINBINDING ASSAYS. Editors W.D. Odell and W.H. Daughaday. Lippincott C ° . Philadelphia and Toronto. 1971, p. 164.

STEROIDS

TABLE

I : Specificity

819

of the antibody

% Cross-reaction Steroids Feb.

Aldosterone Aldosterone-21-monoacetate Aldosterone-18,21-diacetate

72

I00 33 5.40

Apr.

72

I O0

Mar.

73

I00 36 5.0

30 6.0

11 ~ , 1 8 , 2 1 - t r i h y d r o x y - 4 pregnene-3,20-dione Corticosterone Deoxycorticosterone II ~ ,17~ ,21-trihydroxy-l,4pregnadiene-3,20-dione 3 O-hydroxy-5-androsten-17-one Progesterone Testosterone Cortisone 17~ ,21-dihydroxy-4-pregnene3,20-dione Cortisol

% Cross-reaction

0.04 0.015 0.004

0.05 0.0008 0.O04

0.03 0.00002 0.007

0.004 0.004 0.003 0.003 0.002

0.005 0.003 0.00006

O.01 0.004 0.000001

0.0005 0.0005

0.001 0.00003

0.00002 0.000001

pg of aldosterone at B / B o = 0.5 = pg steroid "X" at B/Bo --A O.5 x 100

TABLE 2 : Accuracy

N? of Jeterminations

and precision

Aldosterone Aldosterone measured added (pg) (mean ! SD) (pg)

Percent Coefficient recovery of variation

9

50

47 +

6

94

13

4

80

79 +

3

99

4

I0

IO0

106 +

5

106

5

7

200

212 + 25

106

12

4

400

380 + 34

95

9

B20

STE

~

R O 1DS

6

Figure ] Influence of cellulose coupling on the dilui:~.on curves the anclser~, g : i m ~ a n o e o r b e n t ; B : s e p a r a t i o n o f bound from free fractions by me~n~of atmnoniumsulfate precipitation.

of

60 5fl

B ~

A

@

10

D

" 30 " " 60

Figure 2 Bindingof A : io°c ; B : 37°c.

-

tritiated~aldosterone

' by

the

l~ m;

immun¢~sorbent.

IOC ,

•

~A

90

g ~

7o,

B

60. 511

-

2

~

m

V~ -a~

75 'n,,,,(m+,,,4

Fi$ure ) Rei~ale of trit~ated-eidOaterone by tht, iu~nun<,,~orhent &'C (A) and at ]7"C (B). lOO ~, hound was the n i c l v i r v hound ro the i~nolorbent at zero-time.

.r

ST E R O 1D S

60

821

K

50

Z 0, ~

40

Z

30_

U

2O

10

, 100

A ~B

, 200

400

800

PICOGRAM A LDOSTERONE Figure 4 Influence of late addition of the tracer and second incubation at 4°C on the initial slope of the standard-curve. A : one step method ; B : two steps method. (Means and standard deviations from ]O different standard curves). tO00~ Pg

800

60C

4- 9.17' 0 97)

400

200

/

wi~: chron~onral:~

Figure 5 Correlation of aldosterone (x) and without (y) chromatography.

assays with