Antibody binding efficiency of differently labelled steroid hormones

81

Anaiytrca ChunuzaActa, 275 (1993) 81-87 Elsevler Science Pubhshers B V , Amsterdam

Antibody binding efficiency of differently labelled steroid hormones Jozef G De Boever Lkptzrtmentof Obstetms and Gynecology, lhuvemty Hospital, De Rntehan 185, B-9tXM Ghent (BeIgaun)

Fortune Kohen Department of Hormone Research, Weumann In&m&eof hence, IL-76100 Rehovot (Israel)

Eugene Bosmans Eurogenetxs m

B-3980 Tessenderlo (Belgwnd

(Recewed 29th May 1992, rewsed manuscnpt recewed 30th November 1992)

The bmdmg of estradlol labelled unth 3H or with exterual labels k&m1111o1 and horseradish peroxuiase) to anti-estradlol-6-carboxymethyloxune-bovme serum albumm antlbodles was compared The external labels were coupled covalently to the steroid via homologous kxboxymethyloxune or heterologous 3- and 17-henxsuccmate bndges Dtierent factors and condlfions mfluencmg this bmdmg and the slope and shape of the calibration graph were studted, mcludmg the type of label, the hqmd- and solid-phase antibody system, the matlYr effect and general mcubatlon conditions, e g , tie and temperature External homologous labels yielded the most sensltwe cahbratlon graphs and the lowest demon hnuts Large differences m the bmdmg of the label and m the shape of the calibration graph between the Merent antities were observed kizywor& Immunoassay, Radmchermcal methods, Anabohc steroids, Antibody bmdmg efficiency, Chemlhmunescent labels, Enzyme labels, Estra&ol, Steroid hormones

Immunoassays offer convement and mexpenslve means for the screenmg and determmatlon of many different compounds In recent years altematwe labels such as enzymes and fluorogemc and chenulummescent compounds have frequently been used for the determmatlon of steroid hormones m blolo@cal matenals, e g , tissues and body flmds Generally, the bmdmg of uniabelled and trltmm C3H)_labelled steroids to spectic antibodies occurs wth comparable avtdlty However, a 50% dtierence m antibody bmdmg potency of Correspondence to J De Boever, Department of Obstetncs and Gynecology, Umversrty Hospital, De Pmtelaan 185, B9UOO Ghent (Belguun)

estradlol (E,) and [3H]E2 has been described [l] More problems were encountered urlth lodme radlolsotopes as these were coupled to the steroid hgand via a chermcal bridge This was most convemently done usmg the same bridge structure (e g , hermsuocmate or carboxymethyloxune) as used m the steroid-protem conjugate that served as the mummogen [2] However, because antlbodtes tend to recogmze the bndge m addltlon to the sterotd, &me-labelled steroids generally have a higher affimty for the antlhody than does the unlabelled steroid Consequently, the assay may be less sensmve than the cokespondmg- assay usmg 3H-labelled steroid In some mstances the sensltlvlty may even be completely lost [3] The

OCQ3-2670/93/$06 00 Q 1993 - Elsewer Science Pubhshers B V All whts resewed

82

sensltwity can be restored by changmg the structure of the bndge so that the avld1t.y of the antibody for the radlohgand 1s dunmlshed [41 These hapten unmunoassays usmg external labels have been classfied as homologous or heterologous accordmg to whether or not the immunogen and labelled analyte contam an identical bridge [5] Even heterologous-site steroid unmunoassays m which the bridge m the mununogen and 111the labelled analyte 1s attached to dfierent positions on the steroid molecule (e g , C-3 and C-11 of the rmg structure) have been evaluated [4,6] Other external labels, such as the chenulumlnescent compound rsolummol and the enzyme horseradish peroxldase (HRP), have also been coupled to the steroid hormones by means of a chenucal bndge Consequently, the homology and heterology of the bndge and of the site of attachment are nnportant charactenstlcs of these conjugates Indeed, on several occasions it has been shown that sensltlvlty of enzyme mununoassay (EIA) could be considerably mcreased when the steroid denvatlve used for producing the enzyme conjugate and the nnmunogen dtiered shghtly [7-101 On the other hand, with ~solummollabelled steroids the mam problem concemmg increased avldlty of the antibody for the labelled steroid could be overcome by delayed addition of the conjugate [ill In fact, sensltlve chenulmlnescence nnmunoassays (CIAs) for steroid hormones could be elaborated using homologous combmatlons of numunogen and labelled steroid [ 12-151 Homology and heterology of sterold-nnmunogen and steroid-label conjugates can thus sometimes be decmve Wlfh regard to assay sensltmty, whereas on other occasions they are not Moreover, other components of the mununoassay system and the assay condltlons themselves may strongly influence the slope of the cahbratlon graph The antibody type, Its use 111the hqmd or sohd phase, the nature of the solid phase, the label itself, the matrix and the general mcubatlon conditions, e g , tune and temperature, each on Its own or 111combmatlon, may all have a great impact on assay sensitiwty Examples of this are given m this paper In this work, different antibodies to estradlol-17/3 (a polyclonal antlbody

I G DE Boever et al. /Am!

Chtm Acta 275 (1993) 81-87

and two monoclonal an&&es), dtierent labels L3H, lsolummol and the enzyme horseradish peroxldase (HRPII, liquid- and solid-phase systems, homologous and heterologous combmatlons of antibody and steroid-label coqugates and different matrices were stutlled and compared On all occasions incubation conditions were selected so as to obtam optnnum or near-optnnum assay sensltmty and still permit compansons of dtierent assays

EXPERIMENTAL

Chemicals and reagents Steroids, mlcroperoxldase (MP-11, EC 1 11 171, bovme serum albumm (Cohn fraction V) (BSA), Tween 20 and thnnerosal were obtamed from Stgma (St Louis, MO), ethanol, hydrogen peroxide (300 g 1-l solution) and actlvated charcoal from Merck (Darmstadt, Germany), Dextran-T70, Sephadex G-100 and Sepharose-protem A from Pharmacla (Uppsala, Sweden), rmcrotltre plates (Maxtsorp) and sealing tape from Nunc (Kampstrup, Denmark), 3,3’, 5,5’-tetramethylbemdme (TMB) from Boehrmger (Mannhelm, Germany), second antibody, rabbit anti-mouse nnmunoglobulms (code 2259) from Dakopatts (Glostrup, Denmark), donkey antimouse antibody-coated cellulose suspension (anti-mouse Sac-C& from Wellcome (Beckenham, UK) and [2,4,6,7-3H,]estradlol (speclflc actlvlty 110 kc1 mol-I) from Amersham Intematlonal (Amersham, Bucks , UK) Different ConJugates of horseradish peroxldase (HRP) wth estradlol (E,) coupled through carboxymethyloxune (CM01 or hemlsuccmate (HS) spacer were purchased E,3-HS-HRP and E,6-CMO-HRP from Dr A Roda (Umverslty of Bologna, Bologna, Italy) and E,-17-HS-HRP from Sigma The assay buffer for RIA and CIA was sodium phosphate (50 mm01 l-l, pH 8 O), contammg 9 g 1-l NaCl, 100 mg l- 1 BSA and 1 g 1-l sodmm azlde The assay buffer for EIA was sodmm phosphate (25 mm01 l-‘, pH 8 01, contammg 9 g 1-l NaCl, 250 mg 1-l BSA, 125 mg I-’ thunerosal and 4 15 g 1-l EDTA

JG De Boever et al /Anal Chm. Acta 275 (1993) 81-87

The coatmg buffer was sodmm carbonate (50 mm01 1-1, pH 9 6) contammg 100 mg I-’ thunerosal The wash solution contamed 9 g I-’ NaCl, 1 g 1-l soduun azlde (CIA) or 100 mg I-’ thnnerosal (EIA)and05mll-‘Tween20 The blockmg buffer for CIA was assay buffer contammg 1 g l- ’ BSA and that for EIA was potassium phosphate (50 mm01 1-l pH 8 0) contaming 1 g 1-l BSA and 100 mg 1-l thnnerosal Specific reagent solutions for CIA and EIA were as described previously [16-181 Dextrancoated charcoal was prepared by mung 0 5 g of activated charcoal with 50 mg of Dextran T70 m 100 ml of assay buffer

A Model 2000 Bmcounter and a Model 2010 automated Blocounter from Lumac Systems (Basle, Swn%zerland) were used for hght measurement Photons generated durmg the chenulununescent reaction were recorded for 10 s and an mtegrated photon count was obtamed The absorbance of the coloured product that resulted from the enzymatic converslon of tetramethylbenzldme was measured at 450 nm usmg an MPR-A4 rmcroplate reader from Eurogenetics (Tessenderlo, Belguun) Procedures

Preparatwn of antdmhes Conjugates of steroid hormones convalently coupled to BSA were syntheslzed as described [191 Polyclonal antI-E,-6CMO-BSA were raised m rabbits 1191and monoclonal anti-E,-6-CMO-BSA, MoAb clones 15 and 2F9, were prepared as descclbed 1201 Synthem of conlugates The chenuhunmescent conJugate E,-6-CMO-ammobutylethyhsol~ol (E,-6-ABEI) was synthesized according to Schroeder et al. [21], as described previously [lo] An E,-6-CMO-HRP conjugate was synthesized by the mured anhydnde method [22], as modified by RaJkowslu et al 1231,and purtied by Sephadex G-100 chromatography, followed by extensive dlalysls Zmmunoassays for estradwZ m serum and uz salrva These were of the &ect type, I e , without prior extraction of the steroid from serum or

83

sahva Instead, the samples were added directly to the mcubatlon nuxture (50 ~1 of serum and 100 ~1 of saliva m a final incubation volume of 200 /.Ll) Radwunmwwassay (ZUA) Volumes of 0 1 ml each of diluted antlbody clone 15 or 2F9, [31-IlE2 and dlsplacmg agents 1241 and a standard m steroid-free serum [17] or a serum sample were mnced and mcubated ovemlght at 4°C Dextrancoated charcoal was used to separate bound and free hgands The antibody-hgand complex 111the supemate was decanted mto 3 ml of lrquld scmtlllatlon cocktall and the radloactimty was counted m a Packard Model 3255 beta counter for 5 mm Chenulum8~scence unmunoassay (CL4) Three systems were studied, as follows A “hqtud” system m test tubes, usmg soluble antisteroid antibody. After incubation, phase separation was effected by the addltlon of second antibody-coated cellulose (Sac-Cell, centhgatlon and recovery of the antibody bound fraction m the pellet A “hquld” system m nucrofitre plates At the start of the assay, soluble antisteroid antlbody was added to the wells, which had been coated m advance with second antibody Durmg mcubatlon for the assay, antisteroid antibody could bmd to second antibody Phase separation was effected by mverslon of the plates A “sohd” system m nucrotltre plates The wells of the plates were coated with second antibody, after which antisteroid antibody was bound to second antibody, all before the immunoassay was started In all CIAs two consecutive mcubatlons (at room temperature) were performed In the first mcubatlon spec& antisteroid antibody was mcubated wth steroid present m the standards or m the samples When the samples were saliva, the standards were dissolved m buffer [16] When the samples were serum, standards were added m steroid-free serum [17], and 111all wells dlsplacmg agents were added A mucture of natural and synthetic steroids was used [24] In the assays mth serum samples a wash concluded the first incubation, then the steroid-lsohumnol conjugate was added In the assays with sahva samples the conjugate was added without a pnor wash step

84

This was followed by the second mcubatlon and subsequently by separation of antibody-bound and -free fractions Treatment of the sterold-lsolummol-contammg fraction and hght measurement have been described [16,17] Enzyme unmunoassay (EL4) Both liquid and solid systems on mlcrotltre plates were studied The liquid system was as described for CIA The solid system was obtamed by either direct coatmg of specific antisteroid antibody to the wells or bmdmg it to a second antibody as m the CIA system [31 The enzyme nnmunoassay was carried out at room temperature m a single mcubatlon The sequence of additions of the reagents, 1 e , steroid, steroid-HRP, buffer or matrix (mcludmg daplacmg agents) did not mfluence the results However, m the hqurd system, the antisteroid antlbody was added last The assay was stopped by washmg the mlcrotltre plate followed by addmg substrate and chromogen solutions 1181 The enzymatic reaction was stopped after 10 mm by adding 2 M HCI The absorbance at 450 nm was recorded

RESULTS AND DISCUSSION

Calibration graphs covermg the range 1 S-50 pg of E, of different assays for estradlol are presented m Figs l-4 The polyclonal antiserum (Fig 1) and the monoclonal antibodies 2F9 (Figs 2 and 3) and 15 (Fig 4) were obtamed by mnnumzatlon with E,-6-CMO-BSA Table 1 ldentlfies the curve numbers m Figs l-4 The polyclonal antibodies (Fig 1) recogmzed and bound well both homologous E,-6-CMOHRP and heterologous E,-3-HS-HRP conjugates m the presence of serum (curves 4-6) They did not, however, bmd the heterologous E,-17HS-HRP coqlugate, either m the presence (curve 7) or m the absence (results not shown) of serum The sensltlvltles at zero bmdmg, 1 e , the 2a hmits of detection of estradlol, were 3 5 pg (curve 51, 2 6 pg (curve 4) and 15 pg (curve 6) Monoclonal antibody 2F9 was used m a variety of types of assay (Fig 2), mcludmg RIA, CIA and EIA m the presence of serum (curves 1, 2, 4 and

J G De Boever et al /Anal Chm Acta 275 (1993) 81-87

PoAB

pg E*added Fig 1 Cabbratloo graphs for enzyme ~unoassays for estradlol, using polyclonal anti-E, -6-CMO-BSA ant&x&es EIAs were of the sohd type on MTP The reactlon rmxture (200 ~1) contamed 50 ~1 of serum and 150 ~1 of assay buffer Incubatlon for 1 h at room temperature

6) and CIA in the presence of saliva (curve 3) Comparison of two “liquid” system CIAs, one in test-tubes with separation of bound and free phases by means of Sac-W (curve 2) and one m nucrotltre plates (curve 31, both usmg the same E,-6-CMO-ABE1 conjugate, revealed that the bmdmg lonetics m rmcrotltre plates were not necessarily slower or less efficxent than wth soluble reagents The RIA (curve 1) was also performed Hnth soluble reagents The mcubatton tune, however, (overmght at 4°C) was much longer than that for the CIA (curve 2) RIA (curve 1) and EIA unth homologous conjugate (curve 4) had acceptable slopes and sensltlvlty, their detection llrmts bemg 5 pg per tube (curve 1) and 1 pg per well (curve 41, respectively However, by far the most sensrtlve assay was the CIA for salivary E, (curve 3) It had a detection llrmt of 0 2 pg per well, equivalent to 3 8 pmol 1-l The bmdmg of heterologous E,-3-HS-HRP to 2F9 (curve 6) was weak the absorbance was 0 3 Hnth no E, added Consequently, the slope of the cahbratlon

J G De &ever et al. /Anal

Chm. Acta 275 (1993) 81-87 MoAB

85

2F9

MoAB

i

15

15

ti

;0

pi 2Qddod Fig 2 Caliiratlon graphs for RIA, CL4 and EIA for estradrol, usmg monoclonal ~~~I-E,-~-CMO-ESA antibody 2F9 The reaction muture contraned 50 ~1 of serum and 150 ~1 of buffer (curves 1,2,4 and 6) or 100 ~1 each of sabva and buffer (curve 3) Incubation conditions curve 1, hqmd, RIA m test-tubes, ovemlght at 4”C, curve 2, bqmd, CL4 m test-tubes (Sac-Gel), 1 and 2 h at room temperature, curve 3, bqmd, CL4 m~,!JOmm+30mm,curves4and6,sohdEIAmMTP,1 h at room temperature

MoAB

2F9

Fu 4 Cabbrabon graphs for RIA, CIA and EIA for estradlol usmg monoclonal ~II~I-E,-~-CMO-ESA antibody 15 The reaction nuxture contamed 200 ~1 of buffer kurve 3) or 50 ~1 of serum and 150 1.11of buffer (curves 1,4,5 and 6) Incubation condrtlons curve 1, hqmd, RIA m test-tubes, overnight at 4%. curve 3, hquld, CL4 m MTP, 2 h+5 mm at room temperature, curves 4-6, bqmd, EIA and MTP, 1 h at room temperature

graph was shalknv The heterologous coqugate E,-17-HS-HRP did not brnd to antibody 2F9 (results not shown) In Fig 3, showmg the relative bmdmg (B/B,) of cowugates by 2F9, the dtierTABLE 1 Immunoassays for estradlol-178

4

l@5 &J pg&added

Fu 3 Caliiratlon graphs for RIA, CIA and EIA for estradd, usmg monoclonal antibody 2F9, expressed as percentage relatwe bmdmg, E/B,, For explanation of curves, see FU 2

Curve Assay Labelled No a type steroid b

Obtamed from b

Sohd phase for separation of bound from free

1 2

RIA CIA

Amersham

DCC

Synthewd

’ Sac-G9

3

CIA

4

EIA

5

EIA

6

EIA

7

EIA

i3HlE, E2-6-CMOAEEI E, -6CMOABE1 Ez -6-CMOHRP E+-CMOHRP E, -3-HSHRP E,-lFHSHRP

a Fw l-4 b See Ckmcals c Synthesized by the authors

Synthesrzed c MTP Roda

MTP

Synthesmzd’

MTP

Roda

Ml-P

Sigma

hITP

a& reagents for more detads

86

ences between the slopes of these cahbratron graphs are apparent The effect of the matrrx on the immunoassay performance 1s rllustrated m Ftg 4 In the CIA (curve 31, mcubatron was carrred out m buffer In RIA and EIA (curves 1 and 4-6),50 ~1 of serum m 200 ~1 of mcubatron volume were present There was a substantial decrease m sensrtrvrty m the presence of serum The detectron hmrts of CIA (curve 3) and RIA (curve 1) were 0.5 pg per well and 8 pg per tube, respectrvely One of the E,-6-CMO-HRP comugates (curve 4) and the heterologous conjugate E,-9HS-HRP (curve 6) bound very weakly to MoAB clone 15 The heterologous E,-17-HS-HRP was not bound (results not shown) From the results, rt 1s apparent that, when setting up an Immunoassay, espeaally one using an alternative, 1e , non-radroactrve label, care must be taken not to dectde heedlessly on the apphcabrhty of either antibodies or labelled hapten for mmmnoassay The best and safest way of checkmg the value of an antrbody 1s strll by using a 3H-labelled hgand Drfferences between a hgand and the trrttated hgand are obvrously not of the kmd to bras results If these results are satrsfactory m terms of trtre and dtsplacement of the label by unlabelled hgand, other, external labels may be tested for bmdmg to the antibody Of the three antibodies descrrbed here the polyclonal one bound E,-HRP connrgates better than drd the monoclonal antibodies 2F9 and 15 However, extensive testing and comparison of results revealed that MoAb 2F9, m combmatron wrth homologous E,-6-CMO-HRP, offered better sensrttvrty, a lower detectron lrmrt and better specrfrcrty than the polyclonal and monoclonal 15 antrbodies It was therefore selected for elaboratmg an EIA for direct measurement of estradrol m serum [181 Although the combination of homologous antrbody and steroid-enzyme label ongmally was Judged less suitable for developmg sensitrve enzyme rmmunoassays for steroid hormones [5,8,10,25], more recent studies mdrcated that rt was possible to develop homologous EIAs for steroid hormones, e g , estradiol, with very good sensrtrvrty [26] This has recently been confumed

J G De Boever et al /AnaL Chun. Acta 275 (1993) 81-87

m several laboratories by the development of sensrtrve EIAs for E, [27-311 In all mstances the homologous combmatron mcluded the use of E,6-CMO-HRP The problem of bndge recognition has not been raised m chermlummescence lmmunoassays The homologous E,-6-CMOABE1 comugate has been used for the development of sensrtrve CIAs for E, m serum and m sahva [24,32] Obvrously the problem of bridge recogmtron and bndge bmdmg resides m the use of todmated estradrol Recently suggesttons were made for practical ways of avordmg these phenomena m estradrol radrounmunoassay [33] The authors thank the Belgran National Fund for Screntrfic Research (NFWO), the Bmatronal Science Foundatron, Jerusalem, and the World Health Orgamzatron for financml support

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10 11 12

13

14 15

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Chm Acta 275 (1993) 81-87

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