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ANALYTICAL

BIOCHEMISTRY

Analytical Biochemistry 366 (2007) 175–181

Development of dual-enzyme-based simultaneous immunoassayfor measurement of progesterone and human chorionic gonadotropin

Anupam Basu a,*,1, Saumen Kumar Maitra b, Tulsidas G. Shrivastav a,2

a Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka New Delhi 110067, Indiab Department of Zoology, Visva Bharati University, Santiniketan 731235, India

Received 6 January 2007Available online 12 April 2007

Abstract

The development of a simultaneous multianalyte immunoassay for the detection of progesterone and human chorionic gonadotropin(hCG) in serum is described. In this simultaneous multianalyte assay, two different enzymes, viz. horse radish peroxidase (HRP) andalkaline phosphatase (ALP), were used as markers. To the simultaneous immobilized progesterone and hCG antibody microwells,50 lL of different concentrations of combined standards or serum samples was added in duplicate and then 100 lL of combined conju-gate reagent, composed of 17-a-OH-P-ALP and hCG-biotin was added to all the wells and incubated for 1 h at 37 �C. After incubation,the contents of the wells were decanted and washed thoroughly with running tap water. After washing, 100 lL alkaline phosphatase sub-strate along with streptavidin–horseradish peroxidase was added to all the wells and incubated for 0.5 h at 37 �C. After incubation, thedeveloped color was measured at 405 nm. The absorbency at this stage provides the result for the progesterone assay. The contents of thewells were decanted and washed. In the next step, 100 lL of tetramethylbenzidene/H2O2 reagent was added to all the wells. After 15 minof incubation, 100 lL of 0.5 M H2SO4 was added to all the wells and the color was read at 450 nm. The absorbency at this stage providesthe result for the hCG assay. Sensitivity of the progesterone and hCG assays were 0.118 ng/ml and 0.124 IU/ml respectively. Intra- andinter assay percentage coefficients of variation ranged from 1.8 to 7.1 and 9.1 to 11.5 for progesterone and from 2.1 to 10.4 and 7.2 to 11.3for hCG. There was good correlation between the discrete and the simultaneous assays. For progesterone assay, R2 was 0.99 and forhCG R2 was also 0.99. The developed dual assay for progesterone and hCG may be useful for the diagnosis of abnormal pregnanciessuch as miscarriages and ectopic pregnancies.� 2007 Elsevier Inc. All rights reserved.

Keywords: Simultaneous assay; Progesterone; Multianalyte; hCG; Immunoassay; Dual assay

Progesterone and hCG3 are the two important hor-mones responsible for maintenance of pregnancy. Proges-terone appears to have multiple functions, the most

0003-2697/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2007.04.015

* Corresponding author. Fax: +505 272 9105.E-mail address: abasu@unm.edu (A. Basu).

1 Present address: Department of Cell Biology and Physiology, Schoolof Medicine, Health Science Center, MSC08 4750, 1, University of NewMexico, Albuquerque, New Mexico NM 87131, USA.

2 For reprint requests E-mail: drtg.srivastav@gmail.com3 Abbreviations used: hCG, human chorionic gonadotropin; BSA,

bovine serum albumin; ALP, alkaline phosphatase; HRP, horseradishperoxidase; TMB, tetramethylbenzidene; PB, phhosphate buffer; PBS,phosphate-buffered saline; NRS, normal rabbit serum; ELISA, enzyme-linked immunosorbent assay.

important of which is preparation of the uterus for theimplantation and maintenance of the pregnancy [1]. Onthe other hand, the major function of human chorionicgonadotropin is ‘‘rescue’’ of corpus luteum during the con-ception cycle for continuous production of progesterone inthe first part of the pregnancy [2]. Normal human repro-duction is a partially successful event with only 22.8% oflive births of conception events [3]. Loss of pregnancy isa physically and emotionally challenging torment.

Existing literature [4,5] indicates that the combined lev-els of serum progesterone and hCG have predictive valuefor the diagnosis and management of abnormal pregnan-cies. Different immunoassays are available for the measure-ment of progesterone and hCG, but they are discrete in

176 Simultaneous dual assay of progestone and hCG / A. Basu et al. / Anal. Biochem. 366 (2007) 175–181

nature. Although in the past 2 decades, simultaneous mul-tianalyte immunoassays for different clinically importantanalytes were developed [6], no single simultaneous multi-analyte immunoassay for quantitative measurement of pro-gesterone and hCG has been reported. Therefore, thepresent study was undertaken to develop a microwell-basedsimultaneous multianalyte immunoassay for the quantita-tive measurement of progesterone and hCG in serum basedon the use of a single solid surface with two different labels.

Materials and methods

Materials

Progesterone, progesterone-3-O-carboxymethyloxime(P-3-O-CMO), 1-ethyl-3-(3-dimethyl-aminopropyl)-carbo-diimide HCL, N-hydroxysuccinimide, bovine serum albu-min (BSA), Freund’s complete adjuvant, diethylaminoethyl Sephadex, human chorionic gonadotropinpolyclonal hCG antiserum (against whole hCG), andsodium azide were purchased from Sigma Chemical Co.(St. Louis, MO, USA). 17-a OH progesterone- 3-O-carbo-xymethyloxime (17-a-OH-P-3-O-CMO) was purchasedfrom Steraloids Inc. (UK). Alkaline phosphatase (ALP;Cat. No. EL-1L), streptavidin–HRP, and TMB/H2O2 wereprocured from Bangalore Genei (India). All other chemi-cals and buffer salts were purchased from Sisco ResearchLaboratory (Bombay, India). The microwell reader usedin this study was from Tecan Spectra.

Buffers

The most frequently used buffer was 10 mM phosphatebuffer saline (10 mM PBS), pH 7.0 (Na2HPO42H2O at0.895 g/L and NaH2PO42H2O at 0.39 g/L) containing0.9% NaCl (10 mM PBS) and 0.1% NaN3. This bufferwas used for coating the microwells.

Microtiter well blocking and stabilizing buffer was 10mM PB containing 0.9% NaC1, 0.2% BSA, 0.1% gelatin,thimerosal, dextran T-70, ethylenediaminetetraaceticacid:dipotassium salt (EDTA:K salt), and 0.01% gentami-cin sulfate.

Conjugate dilution buffer was 50 mM Tris–acetate buf-fer, pH 8.1 (Tris–aminomethene 6.0 g/Lit, 1 mL/L 1 N ace-tic acid (CH3COOH), 5 mM magnesium chloride, and0.5% BSA).

Biological and immunological materials

NRS was collected from New Zealand white rabbits.

Antibodies

Anti-rabbit goat antibody (second antibody). Anti-spe-cies antiserum (as second antibody) was generated in goatusing purified rabbit c-globulin as an immunogen by themethod of Basu et al. [7].

Primary antibodies. Progesterone antibody was gener-ated against P-3-O-CMO-BSA in rabbits according to themethod of Basu et al. [8].

Rabbit polyclonal antibody of hCG against whole hCGmolecule was procured from Sigma Chemical Co. (Cat.No. C 8534; Lot 121 K 4891).

NRS and second antibody were used for immobilizationof progesterone and hCG antibody on microtiter wells.

Preparation of conjugates

Enzyme alkaline phosphatase was conjugated to 17-a-OH-P-3-O-CMO by the method of Basu et al. [8] for pro-gesterone estimation.

Biotin was conjugated with hCG according to themethod of Basu et al. [9] for hCG estimation.

Preparation of combined working conjugate dilution

Alkaline phosphatase conjugated to 17-a OH progester-one (17-a-OH-P-ALP) and biotin conjugated to hCG(hCG-biotin) were combinedly diluted in the 50 mMTris–acetate buffer, pH 8.1.

Immobilization of primary antibody to the microwell

Both progesterone and hCG antiserum were immobi-lized to the microtiter wells, according to the method ofShrivastav et al. [10]. Briefly, 250 lL of (1:1000) NRSdiluted in water was added in each well of the microtiterplates and incubated overnight at 37 �C. Plates werewashed in running tap water and 250 lL of anti-rabbit goatantibody (1:4000) diluted in 10 mmol/L PBS, was added toall the wells of microtiter plates and incubated for 2 hoursat 37 �C. After washing, 100 ll of combined dilutions of P-3-O-CMO-BSA antiserum and hCG antiserum were addedand incubated overnight at 2–8 �C. After incubation, thecontents of wells were decanted and washed under tapwater 10 to 12 times by filling, decanting, and flicking.The blocking and stabilizing buffer was added (250 lL) toall wells, incubated 1 h at 37 �C. After decanting the con-tent, plates were dried and kept under desiccation at 4 �Cfor further use. The optimal dilution of progesterone andhCG antibody and their respective conjugates were workedout by checkerboard assay.

Preparation of combined standard of progesterone and hCG

Horse serum was used for the preparation of combinedstandard. Endogenous steroid from horse serum wasstripped off using activated charcoal. Briefly, we addedactivated charcoal (50 mg/mL) to horse serum, stirred for2 h at 45 �C, and centrifuged at 15000 rpm to remove thecharcoal. Supernatant was collected and passed through0.45-lm membrane filter.

Fifty micrograms lyophilized hCG (from Sigma; CatNo. C 2047; activity 13,000 IU/mg with respect to IRP75/537) was dissolved in 250 lL of stripped horse serum,

Fig. 1. Standard curve of progesterone assay as obtained by simultaneousdual ELISA. (X-axis in log scale and Y-axis in linear scale; plotted by MSExcel).

Simultaneous dual assay of Progesterone and hCG / A. Basu et al. / Anal. Biochem. 366 (2007) 175–181 177

which corresponds to 2600 IU/mL. On the other hand,1000 lL of working stock of progesterone (1 lg/mL) wasdried and reconstituted to 10 mL of stripped horse serum,which corresponds to 100 ng/mL. From the stock of twoseparate dilutions of (P4 and hCG) seven different com-bined standards of P4 and hCG were prepared, which areas follows:

Stock combined standard H. In a vial, add 207.6(�208) lL of horse serum reconstituted hCG (2600 IU/mL) and 7.2 mL of progesterone (100 ng/mL) and makeup the volume to 9 mL with stripped horse serum; the finalconcentrations of P4 and hCG in this dilution are 80 ng/mLand 60 IU/mL, respectively.

Combined standard G. To 5 mL of combined standard Hadd 5 mL of stripped horse serum. The concentrations ofprogesterone and hCG in this combined standard are40 ng/mL and 30 IU/mL, respectively.

Combined standard F. To 5 mL of standard G add 5 mLof stripped horse serum. The concentrations of progester-one and hCG in this combined standard are 20 ng/mLand 15 IU/mL, respectively.

Combined standard E. To 5 mL of standard F add 5 mLof stripped horse serum. The concentrations of progester-one and hCG in this combined standard are 10 ng/mLand 7.5 IU/mL, respectively.

Combined standard D. To 5 mL of standard E add 5 mLof stripped horse serum. The concentrations of progester-one and hCG in this combined standard are 5 ng/mL and3.75 IU/mL, respectively.

Combined standard C. To 1 mL of standard D add 9 mLof stripped horse serum. The concentrations of progester-one and hCG in this combined standard are 0.5 ng/mLand 0.375 IU/mL, respectively.

Combined standard B. To 4 mL of standard C add 5 mLof stripped horse serum. The concentrations of progester-one and hCG in this combined standard are 0.2 ng/mLand 0.15 IU/mL, respectively.

Simultaneous dual enzyme linked immunosorbent assay(Dual ELISA) procedure

To the simultaneous immobilized progesterone andhCG antibody microwells, 50 lL of different concentra-tions of combined standards or serum samples was addedin duplicate and then 100 lL of combined conjugatereagent, composed of 17-a-OH-P-ALP and hCG-biotin,was added to all the wells and incubated for 1 h at 37 �C.After incubation, the contents of the wells were decantedand washed thoroughly with running tap water. Afterwashing, alkaline phosphatase substrate (p-nitrophenylphosphate, 1 mg/ml, in 10 mM ethanoldiamine) along withstreptavidin-HRP (100 lL) was added to all the wells andincubated for 0.5 h at 37 �C. The developed color was readat 405 nm in a Tecan Spectra microtiter plate reader (capa-ble of measuring absorbency of 96 wells within 8 s). Theabsorbency at this stage provides the result for the proges-terone assay. The liquid contents of the wells were decanted

and washed thoroughly with running tap water. In the nextstep, 100 lL of TMB/H2O2 reagent was added to all thewells. After 15 min of incubation, 100 lL of 5 N HCLwas added to all the wells and the color was read at450 nm in the microtiter plate reader. The absorbency atthis stage provides the result for the hCG assay.

Sample collection

Serum samples were collected by vena puncture frompregnant females. The volunteers were in the age groupof 25–30 years. The study was approved by the InstituteEthical committee.

Comparison with separate assay

To determine the correlation between the discrete assayand the simultaneous enzyme immunoassay, progesteroneand hCG concentrations in serum samples of pregnantwomen (n = 17) were measured by this simultaneous dualenzyme immunoassay and earlier reported discrete ELISAs[8,9]. Correlation coefficient between simultaneous assayand discrete assay was calculated and a regression graphwas plotted separately for progesterone and hCG by SPSS.

Statistical analysis and graphical presentation of data

The statistical analysis and graphical presentations ofdata were done by statistical analysis software SPSS andMicrosoft Excel.

Results

Characterization of standard curves

The standard curves for progesterone and hCG asobtained are shown in Figs. 1 and 2. The slope (m) andintercept (c) of standard curves for progesterone were�1.3 and 0.79 and for hCG were �1.5 and 0.58,respectively.

0

1

2

3

4

5

6

7

0 5 10 15 20 25 30

Bound progesterone(nMOL)

Bou

nd /

Fre

e

Ka = 0.24 x 108 L/mol

Fig. 3. Determination of affinity constant of progesterone antibody asobtained by the simultaneous dual enzyme immunoassay by means ofScatchard plot. (Plotted by MS Excel).

2.5

8

Fig. 2. Standard curve of hCG assay as obtained by simultaneous dualELISA. (X-axis in log scale and Y-axis in linear scale; plotted by MSExcel).

178 Simultaneous dual assay of progestone and hCG / A. Basu et al. / Anal. Biochem. 366 (2007) 175–181

Y ¼ �1:3X þ 0:79½for progesterone assay�:Y ¼ �1:5X þ 0:58½for hCG assay�:

-1

-0.5

0

0.5

1

1.5

2

0 0.5 1 1.5 2 2.5

Bound hCG (nMOL)

Bou

nd /

Fre

eKa = 1.2 x 10 L/mol

Fig. 4. Determination of affinity constant of hCG antibody as obtained bythe simultaneous dual ELISA by means of Scatchard plot. (Plotted by MSExcel).

Table 1Intra- and interassay percentage coefficients of variation for progesteronevalues from pooled serum by simultaneous dual enzyme immunoassay

Pooled serum Mean (n = 7) (ng/mL) 2 SD CV%

Intraassay variations

Very Low 6.54 0.47 7.1Low 10.22 0.53 5.2Medium 21.1 0.88 4.1High 39.1 0.70 1.8

Interassay variations

Very Low 9.04 0.87 9.6Low 12.46 1.4 11.5Medium 24.25 2.2 9.1High 35.28 3.4 9.8

Assay sensitivity or lower detection limit

The sensitivity of the assay was determined as per themethod of Rodbard [11]. The sensitivity or lower detectionlimit was calculated by the formula ALD = A0–2SD, whereA0 is the mean absorbance at 0 dose, SD is the standarddeviation of 20-fold absorbance at 0 dose, and ALD is theabsorbance at lower detection limit. In the simultaneousassay, 0 standard was put 20 times (n = 20) along withduplicate standard doses. From the mean of the 0 doses(each of progesterone and hCG), twice the SD of the 0 dosewas subtracted, the values obtained were interpolated fromthe standard curves, and the concentration found in thisway was designated sensitivity of the respective assays.Accordingly, sensitivity of the progesterone and hCGassays were 0.118 ng/mL and 0.124 IU/mL, respectively.

Affinities of the antibodies

The affinity constants (Ka) of the progesterone and hCGantibodies toward antigen were estimated by use of Scat-chard plots as shown in Figs. 3 and 4 and were found tobe 0.24 · 108 and 1.2 · 108L/mol, respectively.

Intra and interassay coefficients of variation

Intra- and interassay percentage coefficients of variationranged from 1.8 to 7.1 and 9.1 to 11.5 for progesterone andfrom 2.1 to 10.4 and 7.2 to 11.3 for hCG, respectively(Tables 1 and 2).

Correlation coefficient with discrete assay

Serum samples of pregnant women were estimated bydiscrete progesterone and hCG ELISAs and by simulta-

neous dual ELISA. There was good correlation betweenthe discrete and the simultaneous assay. For progesteroneassay the value of R2 was 0.99 and for hCG R2 was also

Fig. 5. Regression graph of correlation between discrete ELISA andsimultaneous dual ELISA for progesterone. Values of progesteroneobtained by discrete ELISA are plotted in X-axis and values ofprogesterone obtained by simultaneous assay are plotted in Y-axis.(Plotted by SPSS).

Fig. 6. Regression graph of correlation between discrete ELISA andsimultaneous dual ELISA for hCG. Values of hCG obtained by discreteELISA are plotted in X-axis and values of hCG obtained by simultaneousassay are plotted in Y-axis. (Plotted by SPSS).

Table 2Intra- and interassay percentage coefficients of variation for hCG valuesfrom pooled serum by simultaneous dual enzyme immunoassay

Pooled serum Mean (n = 7) (IU/mL) 2 SD CV%

Intraassay variations

Very low 0.72 0.03 4.7Low 2.33 0.24 10.4Medium 3.18 0.12 3.7High 5.97 0.13 2.1

Interassay variations

Very low 0.75 0.07 9.3Low 2.3 0.26 11.3Medium 3.57 0.29 8.1High 6.45 0.47 7.2

Simultaneous dual assay of Progesterone and hCG / A. Basu et al. / Anal. Biochem. 366 (2007) 175–181 179

0.99. The regression graphs are presented in Figs. 5 and 6,respectively. The regression equations are as follows:

Y ðSimultaneous progesterone assayÞ¼ 1:06X ðdiscrete progesterone assayÞ � 2:22 and

Y ðSimultaneous hCG assayÞ¼ 1:06X ðdiscrete hCG assayÞ � 0:17

Discussion

An attempt has been made for the first time to developsimultaneous dual enzyme immunoassay for quantitativemeasurement of progesterone and hCG concurrently fromthe same serum sample. Generally, for the generation ofsimultaneous immunoassays, two different strategies have

been employed: (i) use of spatially separated test zones or(ii) use of different labeling molecules [12]. The measure-ment of progesterone and hCG in the present study wasbased on the second strategy, i.e., use of two differentenzymes as markers utilizing microwells as solid phase.The developed assay is very simple and sensitive and isbased on a competitive inhibition principle. Only 50 lLof serum sample is required to obtain the values of proges-terone and hCG within 2 h. Throughout this study, wehave utilized tap water during washing steps, the presentstudy suggests caution about the use of tap water due toits strong reducing properties because of chlorine or ozonetreatment, which may have implications on immobilizedmolecules, so extra care must be given for the handlingof tap water.

The primary task in the development of the presentsimultaneous dual enzyme immunoassays was to immobi-lize both antibodies at the same time on a single solid phase(i.e., microtiter well). The immuno-bridge technique [10]was used to immobilize two antibodies to the microtiterwells. It was observed that a higher concentration of pri-mary antibody was required for immobilization on themicrowells in the simultaneous assay than in the singleassay. Optimum dilution of progesterone antibody forsimultaneous immunoassay was 1:10,000 compared to1:30,000 for discrete immunoassay. Similarly, optimumdilution of hCG antibody for simultaneous immunoassaywas 1:1000 compared to 1:2000 for discrete immunoassay.In the simultaneous antibody immobilization protocol,rabbit polyclonal antibodies of progesterone and hCGhave been used. The high concentration of antibodyrequirement for simultaneous immobilization may be dueto the competition between these two antibodies for being

180 Simultaneous dual assay of progestone and hCG / A. Basu et al. / Anal. Biochem. 366 (2007) 175–181

immobilized immunochemically to the microwells throughanti-rabbit goat antibody. In the first step of the immuno-bridge immobilization procedure, normal rabbit serum wasdiluted with water; the c-globulin (IgG) present in theserum becomes hydrophobic in nature (tends to precipitateout) due to its insoluble nature in water and becomesimmobilized onto the solid surface. In the second step, buf-fer-diluted secondary antiserum was added; the anti-speciesantibody present in the antiserum immobilized immuno-chemically onto the solid surface through immobilized nor-mal c-globulin. Finally, buffer-diluted rabbit polyclonalantibodies of progesterone and hCG were added; anti-gen-specific antibodies present in the antiserum wereimmunochemically immobilized onto the anti-species sec-ondary antibody. The anti-species-specific secondary anti-body forms the immunochemical bridge betweennonimmunized c-globulin and antigen-specific primaryantibodies.

Combined standards of progesterone and hCG havebeen prepared in the steroid-stripped horse serum insteadof in the pooled human serum for the simultaneous dualenzyme immunoassay. This is because human gonadotro-pin antibody does not have any cross-reaction with horsegonadotropin. Earlier Akman et al. [13] used bovine serumfor the development of LH immunoassay. However, hCGantibody had very low cross-reaction with h-LH, h-FSH,and h-TSH.

In the present study, progesterone was conjugated withenzyme ALP and hCG was conjugated with biotin formaking nonisotopic conjugates. The streptavidin-conju-gated HRP was used as marker for biotin-conjugatedhCG. Blake et al. [14] for the first time developed simulta-neous immunoassay using enzyme combination of ALPand b-galactosidase. Other investigators also used the samecombination of enzymes for development of simultaneousenzyme immunoassay [15,16]. The enzyme combinationALP and HRP was also used for development of simulta-neous immunoassay [17,18] but the ready-to-use combinedenzyme conjugate preparations are not mentioned. One ofthe major drawbacks in preparing ready-to-use combinedenzyme conjugates using these two enzymes in the simulta-neous assay is their stability and substrate reactivity at dif-ferent pH. Generally, HRP is stable and reactive in acidicpH, whereas ALP is stable and reactive in alkaline pH.Therefore, it is difficult to prepare ready-to-use combinedenzyme conjugates either in acidic or in alkaline pH. Toovercome this problem in the present work, hCG was con-jugated to biotin instead of HRP. Therefore, it was possibleto prepare ready-to-use combined working conjugatereagents consisting of ALP- and biotin- conjugated proges-terone and hCG, respectively.

Generally, a strong acidic or basic or chelating agentsolution is added to stop enzyme reaction before colormeasurement in ELISA. To stop the ALP-mediated reac-tion, strong basic solution/EDTA is used. In the presentstudy, the ALP-mediated substrate reaction was read with-out the addition of stop solution. This was because the

ALP-mediated reaction kinetics is slow and the measure-ment of color was done by an automated microplate reader(which measures the absorbance of all the wells of the platewithin 8 s) and there was no well-to-well measurement var-iation. By doing so two things have been accomplished:avoidance of an extra step and prevention of reduction inHRP activity due to exposure of highly alkaline pH/EDTA.

Among abnormal pregnancies, the incidence of ectopicpregnancy is increasing and accounts for about 9% of preg-nancy-related mortality [19,20]. Abdominal pain or vaginalbleeding are the only clinical signs and symptoms of ecto-pic pregnancy in the first trimester, though they are notspecific [21]. The diagnosis of ectopic pregnancy is compli-cated by the wide spectrum of clinical presentations, andthe accuracy of initial clinical evaluation is less than 50%[22]. Sometimes the presence of a uterine pseudosac in ultr-asonographic images complicates the interpretation ofsonographic findings [23–25]; hence, additional biochemi-cal parameters are required for the confirmation of ectopicpregnancy [20]. As per available records, a single progester-one value of less than 15 ng/mL with an inappropriate dou-bling time of hCG are considered strong diagnosticparameters for ectopic pregnancy [5]. Thus a combinationof serum progesterone and hCG measurement providesbiochemical information for early diagnosis of abnormal/ectopic pregnancy [26]. The developed simultaneousELISA for serum progesterone and hCG may be usefulto rule out the presence of abnormal pregnancies afterthe notice of conception. This test may act synergisticallywith ultrasonographic tests for the diagnosis of differentabnormal pregnancies. For lack of availability of sufficientsamples from ectopic patients and miscarriage subjects, wewere unable to check the developed ELISA with such sam-ples. In future, the developed dual ELISA can be testedwith samples from abnormal pregnancies.

Acknowledgments

The authors are grateful to Head, Department ofReproductive Biomedicine, Professor K. Kalivani, andProfessor Somnath Roy for their valuable suggestionsthroughout the study. Technical support from Mrs. S. Balais also gratefully acknowledged.

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