CLIN. CHEM.

31/10, 1664-1668 (1985)

Chemiluminescence Immunoassay Plasma Progesterone, ith of w Progesterone-Acridinium Ester Used As the LabeledAntigen
A. P. RIchardson, J. B. Kim,2 G. J. Barnard,2W. P. Collins,2 and F. McCapra3

This simple solid-phase chemiluminescence immunoassay

for measurement of progesterone in extracts of venous plasmahas sensitivity and precisionsimilarto thatof conventionalradioimmunoassaywith use of a tritiatedantigen.The
labeled antigen, ii a-progesteryl-2-succinoyltyramine-4-(i 0methyl)..acridinium-9-carboxylate, and a monoclonal antibody to progesterone-il a-succinyl--bovine serum albumin

are incubatedwith a 100-FL aliquot of plasma extract (equivalent to 20 L of plasma) and 50 jL of a suspension of an
lgG fraction of a donkey antiserum to mouse immunoglob-

ulins, covalently attached to cellulose particles. After the antibody-bindingreaction (60 mm at 4 #{176}C), of phos1 mL phate bufferis added to each tube, the tubes are centrifuged
(5 mm, 1500 x g), and the supernatant fluid is aspirated. The washing step is repeated and diluted hydrochloric acid (50 mmol/L, 50 L) is added to the pellet. Luminescence is initiated by oxidation with dilute sodium hydroxide/hydrogen

peroxide.The signal is integratedover 10 S. The lightyield is inverselyproportionalto the progesteroneconcentrationin the standard or sample. AdditIonalKeyphrases:monoclonalantibodies
acridiniumderivatives infertility, ovulation Many methods nonisotopicimmunoassay synthesisof functional

based upon the principles of radioimhave been applied to determination of progesterone peripheral venous plasma from women. In in particular, measurement of this hormone has become a routine procedure formonitoring the treatment of functional infertility (1) and detecting ovulation (2). The increasing availability and usefulness of RIA, however, has raised several problems, discussed elsewhere (3). The several approaches suggested to overcome the disadvantages of RIA, while retaining the specificity and sensitivity of the anti. body-antigen reaction (4), include the use of chemiluminescent markers (5-7). These methods have been shown to be comparable in specificity, precision, and accuracy to established RIAs. In particular, the use of derivatives of isoluminol as labels in immunoassay has become firmly established, and the range of chemiluminescence immunoassays (CIAs) for haptens and proteins routinely determined by this approach is increasing (8). The advantages of isoluminol derivatives (6) include the stability of the labeled conjugates (for years) and the sensitivity with which they may be detected (<10- moliL), although, owing to certain limitations to their use, this sensitivity is not always achieved.
munoassay (RIA)

The chemistry of luminol and its derivatives has been studied since 1928,but themechanism of its chemiluminescence reaction, especially in aqueous conditions, still remains obscure (9). In particular, isoluminol conjugates have a relatively low quantum yield, -1.1% (10). For high sensitivity to be achieved, the actual light measurement must be preceded by an incubation with dilute (2 or 5 mmolJL) sodium hydroxide at high temperature (8). This procedure facilitates the dissociation of the antibody-bound complex and may release the labeled antigen by alkaline hydrolysis. Hence, conditions for each luminescent reaction must be optimized: the temperature and durationof the incubation and the concentration of sodium hydroxide used. Consequently, the speed and convenience of the assay procedure is limited. Recently, acridinium salts and related compeunds have been proposed as alternative chemiluminescent labels (11) because of their higherquantum yield (2-5%) and longshelf life, and because their reaction mechanisms are well understood (12). For example, Weeks et al. have described the use of an acridinium derivative covalently bound to protein in the development of two-site immunochemiluminometric assays formeasurement of a-fetoprotein (13) and thyrotropin (14) in peripheral plasma. Here, we describe the synthesis, properties, and application of a novel hapten-acridinium ester conjugate, lla-progesteryl-2-succinoyltyrainine-4-(10. methyl)-acridimum-9-carboxylate, in the development of a solid-phase CIA for measurement of progesterone in extracts of peripheral plasma from nonpregnant women.

Materials and Methods

Reagents
Tert-butyl carbazate, tyramine, acridine, dicyclohexylcarbodiimide, methyl iodide,nd succinicnhydride were from a a Aldrich Chemical Co. Ltd., The Old Brick Yard, Gillingham, Dorset, U.K. Progesterone, lla.hydroxyprogesterone, Nhydroxysuccinimide, and bovine serum albumin (Cohn Fraction V) were from Sigma Chemical Co., Ltd., Fancy Road, Poole, Dorset, U.K. All other reagents were from BDH Chemicals Ltd., Baird Road, Enfield, Middlesex, U.K. The assay buffer (0.1 molJL, pH 6.0) was prepared by dissolving 12.8 g of sodium dihydrogen orthophosphate . 2H2O plus 2.8 g of sodium hydrogen orthophosphate in 1 L of doubly distilled water containing 1 g of bovine serum albumin and 9 g of sodium chloride.

Antibodies
Ascitic fluid containing monoclonal antibodies to progesterone-ila-succinyl--bovine serum albumin, prepared according to the method ofFantl et al.(15), was a gift ofJ. Coley (Unilever Research, Colworth House, Colworth, Bucks, U.K.). Donkey anti-mouse IgG covalently linked to cellulose particles (Sac-eel; RD 73) was from Weilcome Diagnostics, Temple Hill, Dartford, Kent, U.K.

School of Chemistry and Molecular Sciences, University of Sussex, Falmer BN1 9Q,J, U.K. 2Diagnostic Research Unit, Department of Obstetrics and Gynaecology, Kings College School of Medicine, Denmark Hill, London SE5 8RX, U.K. 3Address correspondence to this author. Received October 3, 1984; accepted July 10, 1985.
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CLINICALCHEMISTRY, Vol. 31, No. 10, 1985

Preparation of Chemiluminescent Marker Conjugate (see Figure 1)

Tert-butyl carbazate was treated with sodium nitrite according to themethod ofCarpino etal. (16) to produce tertbutoxycarbonyl azide. The azide (25 mmol, 3.6 g) was reacted with tyramine (20 mmol, 2.7 g) in 30 mL of anhydrous pyridine at room temperature for20 h. Excess solvent was evaporated and the 2-(tert-butoxycarbonyl)tyramine (compound I) was recrystallized from benzene/pet. ether(60-SO #{176}C fraction) as fine white crystals (14 mmol, 3.3 g, 70% yield, mp 72-73 #{176}C).infraredand nuclear The magnetic resonance spectra were consistent with the structure ofthe product. Analysis gave: C 65.50%, H 8.32%, N 5.90% (C13H19N03 requires C 65.79%, H 8.09%, N 5.90%). Acridine-9-carboxylic acid, prepared from acridine by the method of Lehmstedt and Wirth(17), was added to a large excess of redistilled thionyl chloride. The mixture was refiuxed under anhydrous conditions for 4 h. The liquid was removed by evaporation, leaving a bright yellow powder, acridine-9-carbonyl chloride, which had a characteristic infrared absorbance at 1780 cm1 and could be used without further purification. The chloride (15 mmol, 3.6 g) was suspended in 20 mL of anhydrous pyridine and stirred for 10 mm. Compound I (13 mmol, 3.1 g), dissolved in 30 mL of

III

ProgHS, NI4S

DCC

(III)

pyridine, was added. After 30 mm the solution had become clear and the reaction was allowed to proceed for 20 h at room temperature. The resulting brown solution was slowly poured into 250 mL of iced water, yielding a buff-colored precipitate, which was filtered, washed with water, and dried. Recrystallization from benzene/cyclohexane gave a light-brown microcrystalline product, tert-butoxycarbonyl2-aminoethylphenylacridine-9-carboxylate, Compound II (12.5 mmol, 5.5 g, 85% yield, mp 174-176 #{176}C). infrared The and nuclear magnetic resonance spectra were consistent with the product structure. Analysis gave: C 73.66%, H 6.00%, N 6.00% (CHN2O4 requires C 73.27%, H 5.93%, N 6.33%). Compound 11(10 mmol, 4.4 g) was suspended in50 mL of HBr/acetic acid (450 g/L) and the mixture was stirred for 24 hat room temperature. The resulting yellow suspension was poured into 100 mL of cold water and the pH ofthe solution was adjusted to 7 with 10 molJL sodium hydroxide solution. The resulting precipitate was filtered, washed with water, and dried. Recrystallization from acetonitrile gave (2aminoethyl)-4-phenylacridine-9-carboxylate (Compound III) as a brown powder (5 mmol, 1.7 g, yield 50%, mp 166168 #{176}C). Analysis by mass spectrometry gave M at 342 (CH18N2O2 = 342). lla-Hydroxyprogesterone hemisuccinate was produced from lla-hydroxyprogesterone by the method of Allen and Redshaw (18). This material (0.30 mmol, 130 mg) and Nhydroxysuccinimide (0.33 mmol, 40 mg) were added to a solution of dicyclohexylcarbodiimide (0.39 mmol, 80mg) in 5 mL of anhydrous dimethylformamide, with stirring, at -20 #{176}C. reaction temperature The was allowed to rise to 25#{176}C, stirring was continued for a further 16 h. We and added 20 iL of acetic acid, to consume unreacted diimide, and filtered the solution. Compound 111(0.30 mmol, 100mg) was added to the filtrate, along with enough 0.5 mol/L sodium hydrogen carbonate solution to give a nominal pH of 8.5. The mixture was stirred for another 20 h, then 25 mL of water was added.The products were extracted into chloroform (3 x 20 mL), which was washed with water, dehydrated with anhydrous magnesium sulfate, and evaperated to give a brown oil. This was dissolved in 2 mL of chloroform and applied to the inner edge of a spinning thin-layer chromatography plate. The product,lla-progesteryl-2-succinoyltyramine-4-acridine-9-carboxylate (Compound IV), was eluted with ethyl acetate as a mobile, blue-fluorescent band with Rf 0.45 (silica/chloroform). The observed impurities had an R of <0.2. Evaporation gave a pale-brown solid (160 tniol, 120 mg, yield 45%, mp 122-125 #{176}C). nuclear The magnetic resonance spectrum was consistent with the expected structure, and mass spectrometry gave M at 754 (C47HN2O7 = 754). Compound IV (27 mol, 20 mg) was suspended in 2 mL of methyl iodide in a thick-walled tube, which was cooled in liquid nitrogen and sealed in vacuo. The tube was heated in an oil bath at 100#{176}C 20 h, and the contents were then for refrozen before opening. The reagent was evaporated, leaving a dark, powdery sample of lla-progesteryl-2-succinoyltyramine-4-(10-methyl)-acridinium-9-carboxylate (Compound V, 22 mol, 20 mg, yield 81%, mp 126-130 #{176}C). The nuclear magnetic resonance spectrum was consistent with the proposed structure, and mass spectrometry gave M at 770 (CHN2O7 = 770).

Iv

Sample Collection and Extraction

Blood sampled by vempuncture from healthy, nonpregnant women was transferred into tubes containing lithium heparin as anticoagulant. After centrifugation, the plasma was removed and stored at -20 #{176}C untilrequired. SubseCLINICALCHEMISTRY, Vol. 31, No. 10, 1985
1665

Fig. 1. Synthesis of 11a-progesteryl-2.succsnoyftyramine-4-(10-meth. yl)-acridmnium-9-carboxylate

quently, 100 uL of thawed plasma was extracted with 1 mL of diethyl ether. The aqueous phase was frozen in solid C02/ethanol, and the organic phase was transferred to a second tube and evaporated. The residuewas reconstituted with 500 pL of phosphate buffer. The mean recovery of tritiated progesteronefrom 20 samples was 91.2% (SD 2.1%). In practice we applied no correction factor for experimental losses.

Immunoassay Procedure
(a) We added 100 M.Lof standard solution (over the range 3.9 to 250 pg/tube) or plasma extract, in duplicate, to the assay tubes according to the protocol shown in Table 1. (b) We added 100 uL of the acridinium-progesterone

(100 pgIlOO L of buffer), 100 1zL of monoclonal solution (1/250 000 dilution in buffer), and 50 iL of donkey anti-mouse IgG covalentlyattached to cellulose particles in suspension (Sac-Cel) according to theprotocol in Table 1.The contents ofeach tube were mixed and incubated for 60 mm at 4#{176}C. solid-phase The second antibody sediments rapidly, and it is essential to keep the stock uniformly suspended during addition by miring gently on a magnetic stirrer. (c) We added 1 mL of buffer to each tube (with the exceptionof the total-counts tubes); the contentswere centrifuged immediately (1500 x g, 5 mm) and the superrates were decanted. (d) The pellets (antibody-bound fraction) were washed once by adding 1 mL of doubly distilled water (with the exception of the total-counts tubes), followed by immediate centrifugation as before. The supemates were decanted, and excess fluid was removed by blotting the end of the tube with absorbant paper. (e) We added 50 j.iL 50 mmol/L hydrochloric of acid solution to each tube, including thetotal-counts tubes. After 5 mm the tubes were placed in an LKB 1250 Lummnometer and chemiluminescence was initiatedy rapidlyinjecting b 300 L of 0.2 mol/L sodium hydroxide solution containing 3 mL of hydrogen peroxide solution per liter. The signal was integrated by the machine over a periodof 10 s.A single assay run comprising 100 tubes can be completed within 3 h.
antibody

conjugate

mum dilution was assessed to be 250 000-fold (atthisvalue, 70% of the labelwas bound in the zerotubes and 20% was bound in the tubes containing 250 pg of progesterone). Figure 2 shows the calibration curve (n = 12, mean SD) at this dilution. Sensitivity. The sensitivity ofthe method was determined by calculating the minimum amount of progesterone that could be significantly distinguished from zero (mean binding at zerodose minus 2 x SD). This value, calculated from three calibration curves prepared in triplicate, was 4 (SD 0.1) pg/tube, which is equivalent to 0.64 (SD 0.02) nmol/L. Accuracy. Increasing amounts ofauthenticprogesterone were added to serum of low progesterone concentration (from men), which had been analyzed previously RIA, by and the samples were re-analyzed CIA, with use ofthe by acridinium conjugate. The results (Table 2) show that the bias ranged from -11.9% to -24.1%. If the values were corrected forthe mean analytical recovery ofthe tritiated internal standard, the bias ranged from -2.2% to -9.5% over the range 50.0 to 5.0 nmol/L. Imprecision. A preliminary estimate ofintra-assay variationwas obtained by analyzing 10 replicate samples from three serum pools within a single assay. The corresponding valuesforinterassay variation came from the measurement of progesterone from the three pools conducted in five completely separate assays. The results are given in Table3.

100#{149}

80

60

Calculation of Results
The unknown values were derivedfrom the calibration curves (% B/B0 vs concentration of progesterone in picograms per tube) and multiplied by 0.16 to obtain theresults in nanomoles per liter (the molecular mass of progesterone is 314 g/mol).

40

20

Evaluation of the Assay

Dilution of antibodies. We determined the optimum dilution of ascitic fluid containing monoclonal anti-progesterone antibodies by preparing serial dilutions (range, 20000- to 640 000-fold) of antibody in phosphate buffer. Subsequently, 100 L of acridinium-progesterone conjugate (100 pg per 100 1zL of buffer) was incubated in the presence (250 pg/tube) and absence of authentic progesterone. The opti40

20

10

25

13

06
nmoyt

0 Progesterene

Fig. 2. Calibration curve for progesterone = 22.2 counts in 10 8 (U(.B 1250 Luminometer)

Table 1. Protocol for Progesterone Chemilu mlnescence Immunoassay

Solid Buffer Std.or

Table 2. Assay Results Where Progesteron e Was Added to Plasma (Normally ContaIning 0.1 nmol of Progesterone per Liter)
Label
100 100 100 100 Addsd Observed
value

Antibody
-

ph...

sample
-

Expected value 5.1 20.1

50.1 Mean

Tube Total Std.or sample

-

Volume, pI.
-

Nonspecificinding 200 b Zero 100

-

100 100

50 50 50

5.0 20.0
50.0

nmol/L 4.2 14.6

446

Recovery, % 82.4 72.6

89.0 81.3

Bias, %

-24.1 -29.2
-11.9 -18.7

100

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Table 3. WIthin- and Between-Batch Variation in Plasma Progesterone Measurement

Mean conc

Sample
Within batch Pool 1

nmol/L(andSD)
5.1 (0.4)

CV.

Pool 2 Pool3
Between batChb

20.0(1.9)
51.0 (6.4)

relatively convenient, still highly sensitive. yet The detection limit forthe conjugate has been determined in terms of the minimum amount that could be distinguished from a zero blank (19). Using reaction conditions comparable to those given for the inimunoassay (the measurements were made in an LKB 1251 luminometer), we achieved theresults giveninFigure4.The graph shows the amount that gave a larger signal ( SD) than the zero tube ( SD), 5 x 10#{176} mol/tube. An important feature ofthechemistry ofacridinium salts is the presence oftheir pseudobases in aqueous solutions at apH exceeding7.0(20). This is due to an acid/base mediated equilibrium (see Figure 5), and so it may be reversed by lowering the pH. However, the pseudobase species is prone to decomposition withoutlight emission, so it is necessary to avoidalkaline conditions during storage, handling, and use prior to measurement. To avoidthis effect, we performed the assays in a phosphate buffer (0.1 moIJL, pH 6) without apparent loss ofantibody binding or lightemission. A major advantage ofchemiluminescent labels forimmunoassays is that they are highly stable and have very long shelf-lives, unlike most radiolabeled materials. Dry samples
minimum

125
23.3

Pool 1
Pool 2

12.4 Pool 3 13.4 10 replIcate determinations,each Induplicate. replicatedeterminations, b5 each In duplicate.

6.0 (1.4) 20.1 (2.5) 55.4 (7.4)

Correlation with RIA

The concentrations of progesterone in 38 extracts of peripheral plasma were determined by both RIA and CIA (Figure 3). In this series of assays, the mean bias by CIA was -8.4% and the coefficient ofcorrelation (r) between the two methods was 0.93.

ClinicalUtility
Concentrations of progesterone as determined by both RIA and CIA in serum obtained from 60 women attending an outpatient clinic were classified according to established reference ranges (22) into follicularand luteal-phase groups. Identical clinical information was derivedfrom the two methods.

DiscussIon
Several acridinium salt derivatives are known that efficiently produce light on addition of dilute alkaline hydrogen peroxide in theabsenceofa catalyst (12). Both thesynthesis of these compounds and the reaction mechanism of the chemiluminescence are well understood. The reaction producesa fluorescer, 10-methyl acridone, in an electronically excited state via a dioxetanone intermediate. As a consequence, in an immunoassay, the chemiluminescent moiety is liberated from the antigen (or antibody) before light emission. This feature should minimi2e any quenching effects due toantibody binding, and itresults in therestorationofa relatively high quantum yield without the need to resort to further incubations. Thus the immunoassay is

-173

-183

-193 log IArin molsi

FIg. 4. Chemiluminescence of the acridinium conjugate Counts SD (n = 4) accumulatedIn 20s (LKB 1251 Lumlnometer)

n= 38

HOG

20

r 0.93

40 50 nmot/L (ARIDINI(11) FIg. 3. Correlation between results by y = 3.3 + 0.82x

RIA(y)andCIA

(.,i)

FIg.5. The actidinium/pseudobase

equilibrium
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CLINICAL CHEMISTRY, Vol. 31, No. 10, 1985

ofboth isoluminol (6) and acridinium salts (21) in conjugate form are known to retain immunological and luminescent activity for at least a year. The progesterone-acridinium label, when dilutedto working concentration in assay buffer, has been shown to be stable forat least 12 days in terms oflight yield; itretains least 90% luminescent i.e., at activity. It can also produce a workable calibration curve after 30 days in solution when the number ofcountsinthe zero tube is 78% of what it was in the zero tube forthe original assay (19). This investigation is still atan early stage, but theresults presented here indicate that the hapten-acridinium ester derivative is a useful alternative to isotopically labeled derivatives for measurement of steroids in concentrated extracts of biological fluids. The assay is relatively simple and inexpensive to perform,and it is reasonably reproducible. Although the measurements have a slight negative bias, they correlate clinically and biometrically with existing methodologies: RIA using a tritiated antigen (22) and CIA using an isoluniinol derivative (23). At present, an impediment to the development of CLAs involving acridinium salts is the absence ofa commercially available derivative. For the labeling ofproteins,he acrit dinium compound described by Weeks et al. (11) would seem to be eminently suitable. Smaller molecules such as steroids and drugs could be conveniently linked to Compound III (with the proviso that the hapten is stable to the alkylation step in the synthesis or can be chemically protected). It is to be hoped that an increasing availability of acridinium compounds will facilitate the introduction ofimmunoassays that are suitable as alternatives to established RIAs and
mMAS.

5 Schroeder HR, Boguslaski RC, Canico RJ, Buckler R. Monitoring specific protein-binding reactions with cheiniluminescence. Methods Enzymol, 57, 424-445 (1978). 6. Simpson JS, Campbell AM, Ryall ME, Woodhead JS. A stable chemiluminescence-labeled antibody for immunological aesay8. Nature (London) 274, 646-647 (1979).
7. Kim JB, Barnard GJ, Collins WP, et al. Measurement of plasma estradiol by solid-phase chemiluminescence immunoassay. Clin Chem 28, 1120-1124 (1982). 8. Barnard GJ, Kim JB, Williams JL. Chemiluminescence immunoassay and immunochemiluminometric assay. In Alternative Immunoa8says, WP Collins, Ed., John Wiley and Sons, Chichester,

U.K., 1984, pp 123-152. 9. McCapra F. Chemiluminescence

Chem 8, 231-277 (1974). 10. Lee J, Seliger JJ. Absolute

of

organic compounds. Prog Org

We gratefully acknowledge the contributions ofmany colleagues to different aspects of this work.The investigators received financial support from the Kings Voluntary Eeaearch Trust, theMedical Research Council, DHSS, SERC, and Wallac Oy, Thrku, FinlancL

References
1. Abraham GE, Maroulis GB, Marshall JR Evaluation of ovulation and corpus luteum function using the measurement of plasma progesterone. Obstet Gynaecol 44, 522-552 (1974). 2. Abdulla V, Diver MJ, Hipkin Id, DavisJC. Plasma progesterone levels as an index of ovulation. Br J Obstet Gynaecol 90, 543-554 (1983). 3. Collins WP, Barnard GJ, Kim JB, et al. Chemiluminaecence immunoassay of plasma steroids and urinary steroid glucuronides. In Immunoassays for Clinical Chemistry. WM Hunter, JE Cone, Eds., Churchill Livingstone, Edinburgh, U.K., 1983, pp 373-379.
4. Collins WP. Immunoassay developments. Bibliogr Reprod, 44,

Al-AS (1984).

spectral sensitivity of phototubes and theapplication tothemeasurement of theabsolute quantum yields of chemiluminescence and bioluminescence. Photochem Photobiol 4, 1015-1027 (1965). 11. Weeks I, Beheshti I, McCapra F, et al. Acridinium esters as high-specific-activity labels in immunoassay. Clin Chem 29, 14741478 (1983). 12. McCapra F. Chemical mechanisms in bioluminescence. Ace Chem Res 9, 201-208 (1976). 13. Weeks 1, Campbell AK, Woodhead JS. Two-site immunochemiluminometric assay (ICMA) for human cr-fetoprotein. Clin Chem 29, 1480-1483 (1983). 14. Weeks I, Sturgess M, Siddle K, et al. A high sensitivity inununochemiluminometric assay for human thyrotropin. Clin Endocrinol 20,489-495(1984). 15. Fantl VE, Wang DY, Whitehead AS. Production and characterisation of a monoclonal antibody to progesterone. J Steroid Biochem 14, 405-407 (1981). 16. Carpino LA, Carpino BA, Growley PJ, et al. Synthesis of tertbutyl azidoformate. Org Synth 44,15-17 (1964). 17. Lehmstedt K, Wirth E. Uber einege MS-Acridin-Derivate. Berichte 61, 2044-2049 (1928). 18. Allen RM, Redshaw MR The useofhomologous heteroloand gous 1-125 radioligands in theradloimmunoassay of progesterone. Steroids 32, 467-486 (1978). 19. Richardson AP. Novel Chemiluminescence Imrnwzoassays. D. Phil. Thesis, University of Sussex, U.K., 1984. 20. Zaklika KA. Reaction Mechanisms of Chemiluminescence. D. Phil. Thesis, University of Sussex, U.K., 1976. 21. Campbell AK, Davies CJ, Hart R, et al. Chemiluminescent labels in immunoassay. J Physiol 306, 3P (1980). Abstract. 22. Yousefnejadian E, Florensa E, Collins WP, et al. Radioimmunoasaay of plasma progesterone. J Steroid Biochem 3,893-901 (1972). 23. Kohen F, Kim JB, Lindner HR, et al. Development of a solidphase chemiluminescence immunoassay for plasma progesterone. Steroids 38,73-89(1981).

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