A direct antigen heterologous enzyme immunoassay for measuring progesterone in serum without using displacer

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stero ids 7 1 ( 2 0 0 6 ) 222230avai lab le at www.sc iencedi rec t .comjourna l homepage: www.e lsev ier .com/ locate /s tero idsA dir emeas itAnupa Kua Departm ly Wb Departma r t i cArticle histoReceived 11 April 2005Received in revised form 22SeptemberAccepted 20Available onKeywords:ELISAAntigen heProgesteronDisplacerme-measuring progesterone in serum is described. Six combinations of antigens andenzyme conjugates were tested; the enzyme conjugate 17-OH-progesterone-3-O-1. InProgesteropromotes tlevels of pmenstrualmatically fpregnancy.nance of prdefect, ectsay techniterone in swhich invoinherent p CorrespoE-mail a0039-128X/$doi:10.10162005October 2005line 15 December 2005terologousecarboxymethyloxime-alkalinephosphatase (17-OH-P-3-CMO-ALP) and the immunogenprogesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) were found tobe best. Fifty microliters of standard or serum sample and 100L of the 17-OH-P-3-CMO-ALP enzyme conjugate were added to the antibody coated wells, and incubated for 1h at37 C. Bound enzyme activity was measured by using p-nitrophenyl phosphate as substrate.The sensitivity of the assaywas 0.11ng/mL, and intra- and inter-assay CVs ranged from 5.1%to 9.6%. The analytical recoverieswere 97105%. The serumprogesterone values obtained bythismethodcorrelatedwellwith thoseobtainedby radioimmunoassay; r=0.97 (n=44).More-over, in this ELISA no displacing agent was used or special means was required to displaceprogesterone from corticosteroid binding globulin (CBG). Serum progesterone concentra-tions of subjects, with histories of recurrent spontaneous abortions were also measured,and correlated well with clinical history. 2005 Elsevier Inc. All rights reserved.troductionne, a C21 steroid secreted by the corpus luteum,he development of the endometrial lining. Serumrogesterone rise during the luteal phase of thecycle. If conception occurs, levels increase dra-rom the end of the rst trimester to term duringBecause progesterone is required for the mainte-egnancy low levels are associatedwith luteal phaseopic gestation, and miscarriage. Many immunoas-ques have been developed for measuring proges-erum. Most of these are radioimmunoassay (RIA),lve the handling of radioactivematerials. Given theroblems therein different non-radioactive methodsnding author. Tel.: +91 11 2616 6441; fax: +91 11 2610 1623.ddress: tgsrivastava@nihfw.org (T.G. Shrivastav).have been developed for measuring progesterone, such aschemiluminescence immunoassays (CLIA) [1], time-resolveduorescence immunoassays (TRFI) [2], uorescence polar-ization immunoassays (FPI) [3], along with enzyme-linkedimmunosorbent assays (ELISA). Although CLIA and TRFIAcompare favorablywith RIA in terms of performance (sensitiv-ity), whereas, instruments required for end product measure-ment are costlier. FPIs of progesterone, on theotherhand, havenot achieved desired levels of sensitivity [3,4]. Thus, the effec-tiveness of these techniques in the ordinary clinical setup islimited. Enzyme immunoassays (EIA) are still one of the mostpopular methods for measuring hormone concentrations.Homologous or site-heterologous EIAs using progesterone-11-hemisuccinate (P-11-HS) as the immunogen and P-11-HS see front matter 2005 Elsevier Inc. All rights reserved./j.steroids.2005.10.006ect antigen heterologous enzymuring progesterone in serum wm Basua, Tulsidas G. Shrivastava,, Saumenent of Reproductive Biomedicine, National Institute of Health and Famient of Zoology, Visva Bharati University, Santiniketan 731235, Indial e i n f ory:a b s t r a c tAn antigen heterologous enzyimmunoassay forhout using displacermar Maitrabelfare, Munirka, New Delhi 110067, Indialinked immunosorbent assay (ELISA) for directlystero ids 7 1 ( 2 0 0 6 ) 222230 223or progesterone-3-O-carboxymethyloxime (P-3-O-CMO) as theenzyme conjugate have been developed [59], but failed toachieve proisomers of Pity 0.16pg/for progestdesired senity achievebody geneterone in coenzyme coderivativesconjugatedgen immobfrom 0.5ngattempts wand enzymtive in termperformancsensitivityGeneralthe serum,used as dibuffer to ding proteinas displaceterone diredrotestosteused in samIn the presdirectly froconjugate bis due toterone, as 8to transcorto our knowassay for thterone by E2. ExThis studycal Commi2.1. MaProgesteronCMO), prog(3-dimethyhydroxysucunds comp(DEAE-sephthe Sigmaprogesteronand otherUK. Alkalinfrom Bangwell platesPvt Ltd. Achased from the Sisco Research Laboratory (SRL), Bombay,India.Preteronteronteroncordamsof diilledof 1hlore we wavateamsin 1slowe wa. Thed wtedd PPreterone phd 17-ing tf, 4mL off thecontal-3-(3DAC-and, the. Onhospningof das vod aginoof dipernrosend tALP0) anassayImmrenerene-3-Otheper sensitivity. Mitsuma et al. [10] used geometric-3-O-CMO, for progesterone assay having sensitiv-mL. Different homologous and heterologous EIAserone (bridge and site) were developed to achievesitivity and specicity [11]. The highest sensitiv-d was 0.25pg/mL with human serum using anti-rated against 7--carboxyethyl-thioether proges-mbination with 6--progesterone-hemisuccinate-njugate. Wu et al. [12] synthesized carboxylic acidof progesterone with different bridge lengths andthemwith ovalbumin, then tested them in an anti-ilized progesterone EIA; the sensitivities ranged/mL to 50ng/mL. In the above-mentioned assays,ere made to nd combinations of immunogense conjugates that led to progesterone EIAs effec-s of both sensitivity and specicity. However, thees of these EIAs were not up to mark in terms of[11,12].ly, for the measurement of steroids directly fromdifferent reagents or combination of reagents weresplacer in enzyme conjugate or sample dilutionisplace the steroid from the specic steroid bind-. Danazol, dexamethasone, and cortisol were usedr in enzyme conjugate buffer formeasuring proges-ctly from serum [1315]. Trichloroacetic acid, dihy-rone, saponin, and methyl isothiazolinone wereple dilution buffer for the same purpose [16,17].ent work, for the measurement of progesteronem the serum no steroid displacer either in enzymeuffer or sample dilution buffer was added. Thisabsence of specic binding globulin for proges-0% of progesterone is bound to albumin and 18%tin and remaining 2% is free. The present study isledge, the rst report of an antigen heterologouse direct measurement of serum levels of proges-IA.perimentalwas conducted according to the Institutional Ethi-ttees norms.terials and methodse, progesterone-3-O-carboxymethyloxime (P-3-O-esterone-11-hemisuccinate (P-11-HS), 1-ethyl-3-l-aminopropyl)-carbodiimide-HCL (EDAC-HCL), N-cinimide (NHS), bovine serum albumin (BSA), Fre-lete adjuvant (FCA), diethyl aminoethyl-sephadexadex), and sodium azide were all purchased fromChemical Company, St. Louis, MO, USA. 17-OH-e-3-O-carboxymethyloxime (17-OH-P-3-O-CMO)steroids were purchased from Steraloids Inc.,e phosphatase (Cat. No. EL-1L) was procuredalore Genei, India. Breakable strip-based micro-were procured from Thermo Labsystems Indiall other chemicals and buffer salts were pur-2.1.1.progesProgesprogesBSA acmilligr400Lof dist40mghydrocmixturmixturto actimilligrsolvedaddedmixturat 4 CdistillegeneraBSA an[18].2.1.2.progesAlkalinHS, anaccordIn briein 50each owater1-ethyride (EmixedbationN2 gasline pcontai875Lture wdialyzeTrisamin 1Lthe su9% sucto it aof the(pH 8.board2.1.3.polystyPolystywith Ping to[19].paration of immunogen and generation ofe antibodye-3-O-carboxymethyloxime (P-3-O-CMO) ande-11-hemisuccinate (P-11-HS) were conjugated toing to the method of Basu et al. [18]. In brief, ten(10mg) of P-3-O-CMO or P-11-HS were dissolved inoxan and 400L of dimethyl formamide. In 200Lwater, 20mg of N-hydroxysuccinimide (NHS) and-ethyl-3-(3-dimethyl aminopropyl) carbodiimideide (EDAC-HCl) were dissolved, and the aqueousas added to the steroid solution. The reactions vortex-mixed and incubated overnight at 4 Cthe COOH group of the steroid. One hundredof bovine serum albumin (BSA) was then dis-00mL of distilled water. The activated steroid wasly to the aqueous solution of BSA. The reactions further vortex-mixed and incubated overnightsteroidBSA conjugate was then dialyzed againstater, and lyophilized. Polyclonal antibodies werein New Zealand white rabbits using P-3-O-CMO--11-HS-BSA immunogens as described elsewhereparation of alkaline phosphatase conjugate withe derivativesosphatase was conjugated to P-3-O-CMO, P-11-OH-progesterone-3-O-CMO (17-OH-P-3-O-CMO)o the method of Basu et al. [18], with modication.g of each progesterone derivative were dissolveddimethyl formamide (DMF) and 50L dioxan. Todissolved steroid derivatives, 100L of distilledining 5mg of N-hydroxysuccinimide and 10mg of-dimethyl aminopropyl) carbodiimide hydrochlo-HCl) were added. The reactionmixturewas vortex-incubated overnight at 4 C. After overnight incu-reaction mixture was dried under a stream ofe hundred and twenty-ve microliters of alka-hatase (supplied in a solution of 50% glycerol,5mM Tris, 5mM MgCl2 and 0.1mM ZnCl2) andistilled water were then added. The reaction mix-rtex-mixed and incubated overnight at 4 C, thenainst 5mM Trisaminomethane buffer (0.6 g ofmethane, 1 g of MgCl2, 20 g sucrose, and 1g NaN3stilled water). The dialysate was centrifuged andatant collected; 50% ethylene glycol (v/v), 1% BSA,and 10% ammonium sulphate (w/v) were addedhe mixture was stored at 30 C. The dilutionsconjugate were prepared in 50mM Tris bufferd working dilution was determined by checker.obilization of progesterone antibodies onwellswells of the micro-titer plate were coated either-CMO antibody or with P-11-HS antibody, accord-immuno-chemical techniques of Shrivastav et al.224 stero ids 7 1 ( 2 0 0 6 ) 2222302.1.4. Checker board assayThe optimal dilutions of antibody and enzyme conjugateswere foun2.1.5. PSix prog2ng/mL,10mM PB2.1.6. SNormal snon-pregyears), noalcoholicClinicathe infertof recurrewere in tcycle leng2.1.7. STo the atrationsdred micconjugateALP) werbated forthe wellsning tapity, 100phosphatwas addbated 30wells wawell readby in-homethod.2.1.8. Senzyme coThere weCMO-BSA(P-3-O-CMpared. Usent combterone asCombinP-11-HSCombinP-3-O-CCombin17-OH-Combingate P-11Combingate P-3-Combingate 17-The above-mentioned six assay combinations were testedin the stancoate, anRathemutufactDterovelss ofs wruacepin fpeaereycletistDPrardntraencalcummregrhe an wmaCod cusiStatisn cosinprogRStandrd ddy a6. Tteroend out by checker board assay [19].reparation of progesterone standardesterone-working standards (0ng/mL, 0.5ng/mL,10ng/mL, 20ng/mL, and 50ng/mL) were prepared inS supplemented with 3% BSA.ample collectionerum samples were collected by venapuncture fromnant female volunteers (having age group of 2035t taking oral contraception. All of them were non-with normal food habit.l samples were collected from patients attendingility clinic of the Institute who have had historiesnt spontaneous pregnancy loss. Most of the patientshe age group of 2530 years and having menstrualth of 2531 days (283).tandard displacement assayntibody-coated wells, 50L of different concen-of standard were added in duplicate. One hun-roliters of the working dilution of the enzyme(P-3-O-CMO-ALP/P-11-HS-ALP/17-OH-P-3-O-CMO-e added to all of the wells, which were then incu-1h at 37 C. After incubation, the contents ofwere decanted, and the wells washed in run-water. Finally, to measure the bound enzyme activ-L of substrate solution (1mg/mL of p-nitrophenyle (PNPP) dissolved in 1% diethanolamine buffer)ed into all of the wells, and they were incu-min at 37 C. The color of the solution in thes measured at 405nm by a Tecan Spectra Micro-er. The progesterone concentration was determineduse developed computer program using logitlogelection of best combination of antiserum andnjugatere two progesterone antibodies generated (P-3-O-and P-11-HS-BSA), and three enzyme conjugatesO-ALP, P-11-HS-ALP, and 17-OH-P-ALP) were pre-ing these antibodies and enzyme conjugates, differ-inations (heterologous and homologous) of proges-say were formulated as follows:ation 1: antibody P-11-HS-BSA and enzyme conjugate-ALP;ation 2: antibody P-11-HS-BSA and enzyme conjugateMO-ALP;ation 3: antibody P-11-HS-BSA and enzyme conjugateP-3-O-CMO-ALP;ation 4: antibody P-3-O-CMO-BSA and enzyme conju--HS-ALP;ation 5: antibody P-3-O-CMO-BSA and enzyme conju-O-CMO-ALP;ation 6: antibody P-3-O-CMO-BSA and enzyme conju-OH-P-3-O-CMO-ALP.Theconjugsitivity2.1.9.RIA ofkit (Imcoatedmanu2.2.progesThe lephaseunteermenstcontrasuredlutealples wtheir cthe sta2.3.2.3.1.Standconceabsorbwere cprogralinear[20]. Tantigeof Feld2.3.2.Methomodel2.3.3.The strelatiodoneuputer3.3.1.The ststandaantiboFigs. 1progesare givdard displacement assay as described above.mbination of antiserum and progesteroneenzyme, that displayed the best afnity, slope of curve, sen-d specicity, was chosen.dioimmunoassay (RIA) of the samplessamples was performed by the Immunotech RIAnotech, France) utilizing a progesterone antibody-be and 125I-labeled progesterone, according to theurers guidelines.etermination of normal reference ranges ofne in the different phases of menstrual cycleof progesterone were measured in the differentthe menstrual cycle of normal volunteers. The vol-ere in the age group of 2535 years having normall history (M/H 282) and were not taking any oraltive pills. The serum progesterone levels were mea-ollicular phase, luteal phase, and also in the mid-k. For determination ofmid-luteal value, blood sam-drawn on days 1822 (depending on the length ofs). The reference range was estimated by applyingical formula of mean2S.D. (95% condence limit).ata analysiseparation of standard curve and afnity constantcurves were prepared using MS Excel package. Thetion was plotted in the X-axis (log scale) and they in the Y-axis. Values of the unknown samples,lated by a in-house developed personal computere written in QBASIC language using the logitlogession method according to the method of Rodbardfnity constant of the progesterone antibody for theas estimated by Scatchard plot according to methodn and Rodbard [21].mparison of ELISA with RIAomparison was tested using Deming Regressionng RIA as gold standard.atistical analysistical analyses like, mean, standard deviations, cor-efcient, and Deming Regression of the data wereg theMicrosoft Excel, Dplot, and the Medcale com-ram.esultsandard curves, sensitivity, and afnityard curves and Scatchard plots of the progesteroneisplacement assays using different combinations ofnd enzyme-conjugated progesterone are shown inhe slopes (m), intercepts (c), and sensitivities of thene assay using each of the different combinationsin Table 1.stero ids 7 1 ( 2 0 0 6 ) 222230 225Fig. 1 Doseresponse curve (a) and Scatchard plot (b) of progesterone ELISA using P-11-HS-BSA antibody and P-11-HS-ALPenzyme conjugate (plotted by MS Excel).Fig. 2 Dos ELISP-3-O-CMO3.2. SpeBased on snations wereactivity owith the P-with C18,(4.5%), 17-whereas crBSA antibojugate wasFig. 3 Dos17-OH-P-eresponse curve (a) and Scatchard plot (b) of progesterone-ALP enzyme conjugate (plotted by MS Excel).cicity of the progesterone antibodyensitivity, afnity, and slope of curve, two combi-re further evaluated for cross-reaction. The cross-f the assay using the P-3-O-CMO-BSA antibody11-HS-ALP enzyme conjugate was less than 0.1%C19, and C21 steroids, except for corticosteroneOH-progesterone (6.5%), and pregnenolone (11%),oss-reactivity of the assay using the P-3-O-CMO-dy with the 17-OH-P-3-O-CMO-ALP enzyme con-less than 0.1% with C18, C19, and C21. steroids,except for(15%).3.3. BesconjugateThe combinOH-P-O-3-best in termof curve. Tcal variableeresponse curve (a) and Scatchard plot (b) of progesterone ELIS3-O-CMO-ALP enzyme conjugate (plotted by MS Excel).A using P-11-HS-BSA antibody and17-OH-progesterone (3.1%) and pregnenolonet combination of antibody and enzymeation, of the P-3-O-CMO-BSA antibody and the 17-CMO-ALP enzyme conjugate appeared to be thes of sensitivity and specicity, afnity, and slopehis combination was further studied for analyti-s like recovery, inter- and intra-assay coefcientsA using P-11-HS-BSA antibody and226 stero ids 7 1 ( 2 0 0 6 ) 222230Fig. 4 Doseresponse curve (a) and Scatchard plot (b) of progesterone ELISA using P-3-O-CMO-BSA antibody withP-3-O-CMO-ALP enzyme conjugate (plotted by MS Excel).Fig. 5 Doseresponse curve (a) and Scatchard plot (b) of progesterone ELISA using P-3-O-CMO-BSA antibody withP-11-HS-ALP enzyme conjugate (plotted by MS Excel).Fig. 6 Doseresponse curve (a) and Scatchard plot (b) of progesterone ELISA using P-3-O-CMO-BSA antibody with17-OH-P-3-CMO-ALP enzyme conjugate (plotted by MS Excel).Table 1 Slope, intercept, and sensitivity of progesterone assay, using different combination of antibodies and enzymeconjugatesNo. of assaycombinationAssay combination Slope (m) and intercept (c) Sensitivity of theassay (ng/mL)m c1 P-11-HS-BSA antibody with P-11-HS-ALP 0.68 1.86 0.382 P-11-HS-BSA antibody with P-3-O-CMO-ALP 1.1 1.4 0.313 P-11-HS-BSA antibody with 17-OH-P-3-O-CMO-ALP 1.5 1.97 0.184 P-3-O-CMO-BSA antibody with P-3-O-CMO-ALP 0.89 1.9 0.45 P-3-O-CMO-BSA antibody with P-11-HS-ALP 1.15 1.7 0.26 P-3-O-CMO-BSA antibody with 17-OH-P-3-O-CMO-ALP 2.2 2.45 0.11stero ids 7 1 ( 2 0 0 6 ) 222230 227Table 2 Recoveries of progesterone from exogenously spiked pooled serumSerum pool no. Progesteroneadded (ng/mL)Progesteroneexpected (ng/mL)Progesteroneobserved (ng/mL)Recovery (%)Pool A 8.6Pool B 5 13.6 13.13 97Pool C 15 23.6 24 102Pool D 35 43.6 45.6 105of variation, establishment of normal values, and correlationcoefcients with RIA.3.4. Validity of the progesterone ELISA using theP-3-O-CMO antibody with the 17-OH-P-3-O-CMO-ALPenzyme con3.4.1. RecTo the threcentrationsing, the corecovery wrecoveries105% (Table3.5. ParSerum fromdiluted inpooled humoped ELISAwith standdilution cuear regress(Fig. 7).3.5.1. AsInter- andmined for tation rangefrom 5.1% t3.5.2. CorForty-onesamples) wELISA andsion analysation (%CVRegressionParrd cd byregrenearon:A) = 1.13X (RIA) 1.03gression graphs are presented in Fig. 8.Determination of normal prole of progesterone innstrual cyclermal ranges of progesterone in the different phases ofnstrual cycle of normal subjects as determined by thet ELISA are given in Table 4.Assay of progesterone in the clinical subjectsrum progesterone levels of 17 clinical subjects, all withes of recurrent pregnancy loss, as measured by theTable 3 nal control samples as estimated by developedELISASample nSample 1Sample 2Sample 3Sample 4jugateoveriese fractions of human pooled serum, different con-of progesterone were added externally. After spik-ncentration of progesterone was measured andas calculated for each fraction of the pool. Theof the three spiked samples ranged from 97% to2).allelismpregnant women of fth week of gestation wasthe ratio of 1:2, 1:4, 1:8, with charcoal strippedan serum, for the study of parallelism of the devel-. Each diluted fractions were run in assay alongard doses. The slope of the standard curve andrve of serum sample on logitlog transformed lin-ion were found as 1.45 and 1.56, respectivelysay precisionintra-assay coefcients of variation were deter-he four internal control samples. Intra-assay vari-d from 5.1% to 9.6%; inter-assay variation rangedo 9.4% (Table 3).relation with RIAhuman serum samples (normal as well clinicalere assayed simultaneously by this progesteroneby the Immunotech RIA kit. The Deming Regres-is was performed. The percent coefcient of vari-) of ELISA was 7.5% and %CV of RIA was 6.8%.analysis of the samples yielded the followingFig. 7 standa(Plottelinearis in liequatiY (ELISThe re3.6.the meThe nothe mepresen3.7.The sehistoriIntra- and inter-assay variation of progesterone of four interumber Intra-assay variationMean progesterone(ng/mL)S.D. (n=8) CV (%)4.25 0.41 9.69.86 0.74 7.514.66 1.18 8.027.18 1.43 5.1allelism of progesterone ELISA. Large line isurve and small line is sample dilution curve.DPLOT software using logitlog followed byssion method. X-axis is in log scale and Y-axisscale.)Inter-assay variationMean progesterone(ng/mL)S.D. (n=8) CV (%)3.79 0.36 9.49.76 0.71 7.114.83 0.77 5.127.55 1.65 5.8228 stero ids 7 1 ( 2 0 0 6 ) 222230Fig. 8 Method comparison by Deming Regression between RIA and ELISA (a) and Residual plotting (b). Values of the RIAplotted in X-axis, whereas values of ELISA plotted in Y-axis. The CV of method X is 6.8% where as CV of method Y is 7.5%(by MedCale software).Table 4 Reference ranges of progesterone in thedifferent phases of menstrual cycle as determined by thedeveloped ELISAPhases of menstrual cycle Normal range of progesterone(mean2S.D.)a (ng/mL)Follicular phase (n=51)b 0.32.3 (1.30.5)aLuteal phase [excludingmid-luteal values] (n=50)b0.719.9 (10.34.8)aMid-luteal value (n=50)b 8.130.1 (19.15.5)aa Denotes mean2S.D.b Data withpresent ELwell with thtrial biopsyphase defe4. DiscussionIn developing the present progesterone ELISA, six combi-nations of antibodies and enzyme conjugates were tested.The combination of the antibody P-3-O-CMO-BSA and theenzyme conjugate 17-OH-P-3-O-CMO-ALP was selected forassay development on the basis of its sensitivity, afnity andspecicity.The developed assay is simple, direct, and convenient touse, as it permits the direct addition of the serum sample into the assay, and it requires only one and a half hours to com-Onlyymeone auenTable 5 lossPatent no1234567891011121314151617a LPD: lutein bracket are sample numbers.ISA are shown in Table 5. These values, correlatee ndings of other clinical tests, such as endome-plete.of enzgestersubseq(wherever available) for the diagnosis of lutealct.substrate iassay was fSerum progesterone levels and endometrial biopsy study of the. Serum progesterone (ng/mL)at 2022 days of cycleEndometrial biopsy: clinical7.9 Early secretary enodmetruimcycle days 16173.6 Early secretary enodmetruim2.6 Early secretary enodmetruim7.1 Early secretary enodmetruim5.4 Early secretary enodmetruim5.6 Early secretary enodmetruim2.0 Early secretary enodmetruim1.7 Early secretary enodmetruim7.3 Early secretary enodmetruim2.0 Early enodmetruim1.0 Early enodmetruim1.8 Early enodmetruim6.8 Early secretary enodmetruim5.4 Early secretary enodmetruim6.4 Early secretary enodmetruim16.8 Mid-secretary enodmetruimcycle days 212218.4 Mid-secretary enodmetruimcycle days 2223al phase defect.50L of standard or serum samples and 100Lconjugate are required, for addition to the pro-ntibody-coated micro-well. After incubation andt washing, another 30min of incubation with thes required to get the result. The sensitivity of theound to be 0.11ng/mL.subjects having history of recurrent pregnancyimpression Remarkhaving LPDaLPDLPDLPDLPDLPDLPDLPDLPDLPDLPDLPDLPDLPDLPDhaving No endometrial defecthaving No endometrial defectstero ids 7 1 ( 2 0 0 6 ) 222230 229Achieving specicity along with sensitivity is a well-recognized problem in progesterone enzyme immunoassaydevelopmebodies andogous assaa sensitivit11-OH-propregnane-3tions haveHS antibodshowed ation withantibody wDas [9] devof 3.8 pg/wand Stabening a senswith 11-O5--pregnaent homoloP-7--carbo(P-3-O-CMOO-CMO-HRcarboxethythe combinBSA with P0.25ng/mL,3,20-dione.conjugateslengths) wemon resonAlthough thsamewas n0.5ng/mL).tivity and hhere, usingP-3-O-CMOconjugate was specicwith C18, Cprogesterongetting betCMO as entives may bgesterone aIn the pusing any sdevelopmeplacing agewere used [and cortisoglobulin (Cterone. Othby proteinsulphonic aat low pH oof these distion, somegen with aare also crmixture of different non-related steroids is also used as dis-placing agents in direct assays of steroids like testosteronetradis-reain dassaa dirremecingbiasin ciortinfree-spele folacepreormogeswerhismids. Lucorptionid-lrogeeatedt ELIal p.ELISmeay hasowlethorr, Pratituhouton or. A.Knt cally aenillermunridin88;25kabaoresvolvimoboi Mlarizll 19nt [22]. To this end, different combinations of anti-enzyme conjugates have been tested. A homol-y using a P-11-HS bridge was developed, havingy of 0.5 pg/well and a 32% cross-reaction withgesterone and a 62.5% cross-reaction with 5--,20-dione [23]. Different heterologous combina-also been tried. The EIA comprised of the P-11-y with the P-11-glucuronide enzyme conjugatesensitivity of 25pg/well, with an 8% cross reac-5--pregnane-3,20-dione [24]. Using the P-11-HSith the P-3-O-CMO enzyme conjugate, Saha andeloped a progesterone assay having a sensitivityell, whereas using the same combination, Munrofeldt [8] had previously developed an assay hav-itivity 0.25pg/well, with a 21.4% cross-reactionH-progesterone, and a 62.5% cross-reaction withne-3,20-dione. Hatzidakis et al. [11] tested differ-gous assays (P-3-O-CMO, P-11-HS, P-6--HS, andxethyl-thioether bridge) and heterologous assays-BSA with P-6--HS-HRP, P-11-HS-BSA with P-3-P, P-6--HS-BSA with P-3-O-CMO-HRP and P-7--l-thioether-BSA with P-6--HS-HRP). Among them,ation of the antibody P-7--carboxethyl-thioether--6--HS-HRP was selected, with a sensitivity ofand a 10% cross-reaction with 5--pregnane-Different progesterone-4-succinimidyl-ovalbuminwith varying length linkers (4, 11, and 18 atomre synthesized and used in different surface plas-ance-based immunoassays (SPR) and ELISAs [12].e sensitivities of these SPR assays were good, theot true for the ELISAs, (sensitivity was greater thanThus progesterone ELISAs with both good sensi-igh specicity have been difcult to design. Butas an antigen heterologous combination of theantibody with the 17-OH-P-3-CMO-ALP enzymee have developed a sensitive (0.11ng/mL) as wellassay, having a cross-reaction of less than 0.1%19, and C21 steroids, with the exception of 17-OH-e (3.1%) and pregnenolone (15%). The reason ofter sensitive and specic assay using 17-OH-P-3-zyme conjugate than other progesterone deriva-e due to differential kinetic interaction with pro-ntibody and this can be further studied.resent study, direct assay was developed withoutteroid displacing or blocking agent. Earlier for thent of direct assays of progesterone, different dis-nts such as danazol, dexamethasone, and cortisol1316]. It is reported that danazol, dexamethasonel, which have afnities for corticosteroid bidingBG), have been used in direct assay for proges-er steroids, e.g. cortisol can be displaced from CBG,binding agents such as 8-anilino-1-naphthalenecid (8-ANS) and salicylate, by proteolytic enzymer by heat treatment. The disadvantage of the useplacing agents is that in their blocking concentra-of them reduce the specic binding of the anti-ntibody. In some cases, these displacing agentsoss-react with the antibody. In several cases aand esof crosagentsof thestudy,measudisplamentwhiletranscrest isis nonavailabof dispThewith nthe prcycleshavingin thedefectties ofproducpeak m[27]. Pof reppresenof lutebiopsyThecic inefcacAcknThe auDirectocere grthrougtributiand Mdifferegratefur e f e r[1] Mimac19[2] Kauinim[3] ChpoBuol. These exogenous steroids are potential sourcesction. The complex mixtures of these displacingirect immunoassay interfere with the performancey and the sensitivity is affected. In the presentect ELISA of progesterone was developed for thent of progesterone in serum, using neither anyagent nor any special means and no measure-was noticed. This may be due to the fact thatrculation only 18% of progesterone is bound withwhere as 80% is bound with serum albumin andform. As the major percentage of the progesteronecically bound with albumin, it may be readilyr antibody during immunoassay without the helpr.sent ELISA correlated well with RIA when testedal and clinical subjects. The normal ranges ofterone in the different phases of the menstruale estimated. Serum samples of clinical subjectstories of recurrent abortions were also assayed-luteal phase for the diagnosis of luteal phaseteal phase defects (LPD) are dened as abnormali-us luteum function with insufcient progesterone[25,26]. As reported in most patients with LPD,uteal progesterone levels remain below 10ng/mLsterone values of the subjects having historiesspontaneous abortions were measured by theSA, and someof the values clearly showed the signshase defect and, later conrmed by endometrialA, presented here, then, is both sensitive and spe-suring serum progesterone concentration and itsbeen validated both analytically and clinically.dgementss are grateful to HOD, RBM, Prof. K. Kalivani, andof. N.K. Sethi, for their support, also express sin-de to Prof. Somnath Roy for his valuable suggestionthe study. We sincerely acknowledge the due con-f Dr. Pikee Saxena, Dr. Saroj Goel, Mr. H.C. Pandey. Mazumder of Institutes Infertility Clinic, in theirpacities. Technical support fromMrs. S. Bala is alsocknowledged.c e sSA, Morton MS, Turkes A. Chemiluminescenceoassay for progesterone in plasma incorporatingium ester labelled antigen. Ann Clin Biochem(Pt. 1):2734.kos SE, Khosravi MJ. Direct time-resolvedcence immunoassay of progesterone in serumng the biotin-streptavidin system and theilized-antibody approach. Clin Chem 1992;38:72530.J, Choi J, Yoon DY, Park J, Eremin SA. Fluorescenceation immunoassay of progesterone. Biol Pharm97;20:30914.230 stero ids 7 1 ( 2 0 0 6 ) 222230[4] Hong JY, Choi MJ. Development of one-step uorescencepolarization immunoassay for progesterone. Biol PharmBull 2002;25:125862.[5] Dray F, Andrieu JM, Renaud F. Enzyme immunoassay ofprogesterone at the picogram level usingbeta-galactosidase as label. Biochim Biophys Acta1975;403:1318.[6] Joyce BG, Wilson DW, Read GF, Riad-Fahmy R. Animproved enzyme immunoassay for progesterone inhuman plasma. Clin Chem 1978;24:2099102.[7] Sauer MJ, Foulkes JA, Cookson AD. Directenzymeimmunoassay of progesterone in bovine milk.Steroids 1981;38:4553.[8] Munro C, Stabenfeldt G. Development of a microtitre plateenzyme immunoassay for the determination ofprogesterone. J Endocrinol 1984;101:419.[9] Saha B, Das C. Development of a highly sensitive enzymelinked immunosorbent assay for human serumprogesterone using penicillinase. J Immunoassay1991;12:391412.[10] Mitsuma M, Kambegawa A, Okinaga S, Arai K. A sensitivebridge heterologous enzyme immunoassay of progesteroneusing geometrical isomers. J Steroid Biochem 1987;28:838.[11] Hatzidakis G, Stefanakis A, Krambovitis E. Comparison ofdifferent antibody-conjugate derivatives for thedevelopment of a sensitive and specic progesteroneassay.[12] Wu Y,progeslinkerssurface2002;67[13] Ratcliff125I-radwith a1982;28[14] Carrierproges(17-alpdisplac[15] BarnarR, et anon-co[16] DechauNew aptime-resolved uoroimmunoassay of progesterone withlabeled analyte. Clin Chem 1988;34:5014.[17] Williams GT, Groskopf WR, Baker HN, Agdeppa DA.Determination of steroids by competitive immunoassay.US Patent (US 6,201,141). Assignee: Abbott Park, IL: AbbottLaboratories; 2001.[18] Basu A, Shrivastav TG, Kariya KP. Preparations of enzymeconjugate through adipic acid dihydrazide as linker andits use in immunoassays. Clin Chem 2003;49:14102.[19] Shrivastav TG, Basu A, Kariya KP. Substitution ofcarbonate buffer by water for IgG immobilization inenzyme linked immunosorbent assay. J ImmunoassayImmunochem 2003;24:191203.[20] Rodbard D. Satistical quality control and routine dataprocessing for radioimmunoassays andimmunoradiometric assays. Clin Chem 1974;20:125570.[21] Feldman H, Rodbard D. In: Odell WD, Daugaday WM,editors. Principles of competitive protein binding assay.Philadelphia: J.B. Lipincott; 1971. p. 158.[22] Grover PK, Odell WD. Specicity of antisera to sexsteroids. I. The effect of substitution and stereochemistry.J Steroid Biochem 1977;8:1216.[23] Wang HX, Stott RA, Thorpe GH, Kricka LJ, Holder G, RuddBT. An enhanced chemiluminescence enzymeimmunoassay for serum progesterone. Steroids1984;44:31728.uerogesnjugzymplicartil 1nes Gsociaman13.ulesciee me89;69ull Mngleid-lecle (ncepJ Reprod Fertil 1993;97:55761.Mitchell J, Cook C, Main L. Evaluation ofterone-ovalbumin conjugates with different lengthin enzyme-linked immunosorbant assay andplasmon resonance-based immunoassay. Steroids:56572.e WA, Corrie JE, Dalziel AH, Macpherson JS. Directioligand assays for serum progesterone comparedssays involving extraction of serum. Clin Chem:13148.e PD, Lee B. Direct radioimmunoassay ofterone in bovine plasma using danazolha-2,4-pregnadien-20-yno(2,3-d)isoxazol-17-ol) as aing agent. Can J Vet Res 1994;58:2303.d G, Osher J, Lichter S, Gayer B, De Boever J, Limorl. The measurement of progesterone in serum by ampetitive idiometric assay. Steroids 1995;60:8249.d H, Bador R, Claustrat F, Desuzinges C, Mallein R.proach to competitive lanthanide immunoassay:[24] SaprcoenapFe[25] Joashu1[26] Sodeth19[27] HsimcycoMJ, Foulkes JA, Worsfold A, Morris BA. Use ofterone 11-glucuronide-alkaline phosphataseate in a sensitive microtiter-plateeimmunoassay of progesterone in milk and itstion to pregnancy testing in dairy cattle. J Reprod986;76:37591.ES, Madrigal-Castro V. Hormonal ndings intion with abnormal corpus luteum function in the: the luteal phase defect. Fertil Steril 1970;21:MR, McLahlan RI, Ek M, et al. Luteal phasency: characterization of reproductive hormones overnustrual cycle. J Clin Endocrinol Metab:80412.GR, Savage PE, Bromhan DR, et al. The value of aserum progesterone measurement in theuteal phase as a criterion of a potentially fertileovulation) derived from treated and untreatedtion cycles. Fertil Steril 1982;37:35560.A direct antigen heterologous enzyme immunoassay for measuring progesterone in serum without using displacerIntroductionExperimentalMaterials and methodsPreparation of immunogen and generation of progesterone antibodyPreparation of alkaline phosphatase conjugate with progesterone derivativesImmobilization of progesterone antibodies on polystyrene wellsChecker board assayPreparation of progesterone standardSample collectionStandard displacement assaySelection of best combination of antiserum and enzyme conjugateRadioimmunoassay (RIA) of the samplesDetermination of normal reference ranges of progesterone in the different phases of menstrual cycleData analysisPreparation of standard curve and affinity constantComparison of ELISA with RIAStatistical analysisResultsStandard curves, sensitivity, and affinitySpecificity of the progesterone antibodyBest combination of antibody and enzyme conjugateValidity of the progesterone ELISA using the P-3-O-CMO antibody with the 17-alphaOH-P-3-O-CMO-ALP enzyme conjugateRecoveriesParallelismAssay precisionCorrelation with RIADetermination of normal profile of progesterone in the menstrual cycleAssay of progesterone in the clinical subjectsDiscussionAcknowledgementsReferences

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