Cross-Linking of 17â-Estradiol to MonoclonalAntibodies by Direct UV Irradiation: Application toan Enzyme Immunometric Assay

Laure Buscarlet, Jacques Grassi, Christophe Creminon, and Philippe Pradelles*

CEA, Laboratoire d’Etudes Radioimmunologiques, SPI/DRM/DSV, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France

Jacqueline Dupret-Carruel and Michel Jolivet

bioMerieux, Departement Immunoassays, Chemin de l’Orme, 69280 Marcy-l’Etoile, France

Stephane Mons

Laboratoire de synthese biorganique, CNRS UMR 7514, Faculte de Pharmacie, 74 route du Rhin, 67401 Illkirch, France

Ultraviolet irradiation was used to cross-link 17â-estradioldirectly to monoclonal anti-17â-estradiol antibodies coatedon 96-well microtiter plates. Cross-linking efficiency wasdirectly correlated with both irradiation energy and wave-length. The best results were obtained at 254 (10 J/cm2,45-min irradiation) and 312 nm (40 J/cm2, 160-minirradiation). The irradiation fully denatured both indi-vidual molecules (i.e., 17â-estradiol and monoclonal anti-17â-estradiol antibody), but 17â-estradiol was at leastpartly protected when immunologically bound to theparatope of the antibody. Four different monoclonal anti-17â-estradiol antibodies yielded positive results, demon-strating that this photo-cross-linking has considerablepotential. We used this original approach to develop a newenzyme immunometric assay of 17â-estradiol based onour previously described immunometric procedure, solid-phase immobilized epitope immunoassay, which useschemical agents to cross-link haptens via amino groupsto specific antibodies. The assay was specific (no cross-reactivity with other natural steroids), precise, and sensi-tive (detection limit of 38 pg/mL in human serum). Itcorrelated well with two competitive commercial immu-noassays when tested on 40 human sera.

Since the pioneering work of Miles and Hales1 and thetheoretical study of Jackson and Ekins,2 noncompetitive immu-noassays (i.e., two-site sandwich immunoassays) have been shownto be superior to competitive immunoassays in terms of sensitivity,precision, kinetics, and working range of analyte. However, two-site sandwich assays appear unsuitable for the measurement ofsmall haptens that have a single epitope or that cannot besimultaneously bound by two antibodies because of steric hin-

drance. To overcome this difficulty, different groups have proposedvarious noncompetitive immunometric formats based on differentapproaches using the following: (a) biotinylated hapten andstreptavidin-coated solid phase in a two-site enzyme immunoassay(EIA) for haptens with an amino group,3 (b) anti-â-type and anti-R-type antibodies in an idiometric assay,4,5 (c) anti-meta-typeantibodies,6 and (d) HPLC through the development of a liquid-phase binding assay.7

Our laboratory has longstanding experience in the develop-ment of EIAs for various molecules using acetylcholinesterase aslabel.8 We have described a new enzyme immunometric assay,the solid-phase immobilized epitope immunoassay (SPIE-IA),9

which measures small haptens with much greater sensitivity thana competitive assay using the same specific antibody and the sameenzyme label. This procedure (Figure 1) uses the same antibodyas capture and tracer molecule and involves four sequential stepssummarized as follows: (step 1) immunological capture of thehapten by a specific monoclonal antibody (mAb) coated on a solidphase (i.e., 96-well microtiter plate); (step 2) covalent immobiliza-tion of the hapten on the solid phase using bifunctional cross-linking reagents; (step 3) release of the linked hapten from theantibody binding site by addition of a dissociating/denaturingagent; (step 4) visualization of the linked hapten by the sameenzyme-labeled specific mAb. This procedure has been success-fully applied to the determination of low-molecular-weight peptidehaptens containing a free amino group such as substance P(1348),10 thyroxine (777),10 endothelin (2492),10 leukotriene C4

(626),11 and a sorbin-derived heptapeptide (739).12 The lack of an

* Corresponding author: (e-mail) philippe.pradelles@cea.fr; (fax) (33) 01 6986 77 49.(1) Miles, L. E. M.; Hales, C. N. Nature 1968, 219, 186-189.(2) Jackson, T. M.; Ekins R. P. J. Immunol. Methods 1986, 87, 13-20.

(3) Ishikawa, E.; Hashida, S.; Kohno, T.; Hirota, K. Clin. Chim. Acta 1990,194, 51-72.

(4) Self, C. H.; Dessi, J. L.; Winger, L. A. Clin. Chem. 1994, 40, 2035-2041.(5) Barnard, G.; Kohen, F. Clin. Chem. 1990, 36, 1945-1950.(6) Mares, A.; De Boever, J.; Osher, J.; Quiroga, S.; Barnard, G.; Kohen, F. J.

Immunol. Methods. 1995, 181, 83-90.(7) Hara, T.; Nakamura, K.; Satomura, S.; Matsuura, S. Anal. Chem. 1994, 66,

351-354.(8) Grassi, J.; Pradelles, P. U.S. patent 5,047,330, 1991.(9) Pradelles, P. U.S. patent 5,476,770, 1995.

Anal. Chem. 1999, 71, 1002-1008

1002 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999 10.1021/ac980870n CCC: $18.00 © 1999 American Chemical SocietyPublished on Web 01/22/1999

amino group on the hapten can sometimes be overcome using aprederivatization step, before performing the SPIE-IA, to introducethis reactive function into the molecule as described for thy-roliberin (362).13 In all these assays, homobifunctional cross-linking reagents that react with primary amino groups, (e.g.,glutaraldehyde or disuccinimidyl suberate) were used for thecovalent immobilization of the hapten on the solid phase.

Recently, we described an SPIE-IA for L-thyroxine using directUV irradiation in the covalent immobilization step14 and theprocedure has thus been named “photo-SPIE-IA”. In this work,we presented data suggesting the involvement of the amino groupof thyroxine in the covalent photo-cross-linking.

Direct photocoupling of unmodified radiolabeled ligands totheir receptors (see references in ref 15) or binding proteins16

has been extensively described in the literature. However, to ourknowledge, there is just one published study of antibody bindingsites using direct UV photocoupling of unmodified hapten (mor-phine) with its specific monoclonal antibodies.17

We therefore investigated the feasibility of using direct UVirradiation in the development of photo-SPIE-IA for other non-peptide haptens devoid of an amino group. We report here, forthe first time, the development of photo-SPIE-IA for 17â-estradiol

(E2). The effects of UV irradiation are detailed, and we analyzedthe sensitivity, precision, specificity, and validity of this assay.Extension of this procedure to other molecules is also considered.

EXPERIMENTAL SECTIONApparatus. SPIE-IA was performed with specialized micro-

titration equipment (washer, dispenser, and plate reader) fromLabsystem (Helsinki, Finland). The 96-well microplates (Maxi-sorp) were from Nunc (Denmark). UV irradiation was performedusing a BIO-SUN irradiator from Vilbert-Lourmat (Marne LaVallee, France) with three different 4 × 20 W UV lamps (254,312 or 365 nm) located ∼10 cm above the 96-well microtiter plateand a microprocessor to program the UV radiant exposure(expressed in J/cm2).

Chemicals. Unless otherwise stated, all reagents were fromSigma (St. Louis, MO). Mesterolone was from Sigma, and othersteroids were from Steraloids Inc. (Wilton, NH). [6,7-3H]-Estradiol(2 TBq/mmol) was from Amersham. I-Block was from Tropix(Bedford, MA). E2 standards diluted in serum and relevant serumsamples were provided by bioMerieux (Marcy l’Etoile, France).Acetylcholinesterase (AChE; EC 3.1.1.7) was purified from electriceel (Electrophorus electricus) by affinity chromatography18 and usedin the G4 form for anti-E2 mAb labeling as described elsewhere.19

AChE activity expressed in terms of Ellman’s units per milliliter(EU/mL) was determined using a mixture (Ellman’s reagent) ofacetylthiocholine (substrate) and dithionitrobenzene (chromogen)as previously described.20

Immunochemicals and Enzyme Labels. Monoclonal anti-E2 antibodies (10G6D6, 3F2B7, 5H2A10, 17E12E5) were kindlyprovided by bioMerieux and produced by conventional hybridiza-tion techniques after immunization of mice by E2-bovine serumalbumin (BSA) conjugates in the 6 position of E2 (bioMerieux,personal communication). These antibodies were purified fromascitic fluid by affinity chromatography on protein A.21 Monoclonalanti-E2 antibody (10G6D6) was labeled by covalent coupling ofFab’ fragments to the maleimido derivative of the G4 form of AChEas previously described19 and used as tracer antibody in the photo-SPIE-IA experiments.

E2-AChE conjugate was obtained using a 6-carboxylmethyl-oxime-E2 derivative (6-CMO-E2) by means of a labelingprocedure described elsewhere for another steroid.22

Photo-SPIE-IA Procedure. Incubations were performed inEIA buffer (0.1 M phosphate buffer, pH 7.4, containing 0.15 MNaCl, 10-3 M EDTA, 0.1% BSA and 0.01% sodium azide) and thewashing buffer comprised 0.01 M phosphate, pH 7, containing0.05% Tween 20. The 96-well microtiter plates were coated withanti-E2 mAbs as described elsewhere10 following a saturating stepusing 0.2% I-Block dissolved in pH 7 phosphate buffer containing0.05% Tween 20. After 18 h of incubation at +4 °C the plates werewashed and stored in EIA buffer. The procedure was routinelyperformed as follows:

(10) Pradelles, P.; Grassi, J.; Creminon, C.; Boutten, B.; Mamas, S. Anal. Chem.1994, 66, 16-22.

(11) Volland, H.; Vulliez Le Normand, B.; Mamas, S.; Grassi, J.; Creminon, C.;Ezan, E.; Pradelles, P. J. Immunol. Methods. 1994, 175, 97-105.

(12) Ezan, E.; Tarrade, T.; Cazenave, C.; Ardouin, T.; Genet, R.; Grassi, J.;Grognet, J. M.; Pradelles, P. Peptides 1995, 16, 449-455.

(13) Etienne, E.; Creminon, C.; Grassi, J.; Grouselle, D.; Roland, J.; Pradelles, P.J. Immunol. Methods 1996, 198, 79-85.

(14) Etienne, E.; Creminon, C.; Lamourette; P.; Grassi, J.; Pradelles, P. Anal.Biochem. 1995, 225, 34-38.

(15) Bouchet, M. J.; Goeldner, M. Photochem. Photobiol. 1997, 65, 195-200.(16) van der Walt, B.; Nikodem, V. M.; Cahnmann, H. J. Proc. Natl. Acad. Sci.

U.S.A. 1982, 79, 3508-3512.(17) Sawada, J.; Yamazaki, T.; Terao, T. Mol. Immunol. 1993, 30, 77-86.

(18) Massoulie, J.; Bon, S. Eur. J. Biochem. 1976, 68, 531-539.(19) Grassi, J.; Frobert, Y.; Pradelles, P.; Chercuitte, F.; Gruaz, D.; Dayer, J. M.;

Poubelle, P. E. J. Immunol. Methods 1989, 123, 193-210.(20) Pradelles, P.; Grassi, J.; Chabardes, D.; Guiso, N. Anal. Chem. 1989, 61,

447-453.(21) Langone, J. J. J. Immunol. Methods 1982, 55, 277-296.(22) Porcheron, P.; Moriniere, M.; Grassi, J.; Pradelles, P. Insect. Biochem. 1989,

19, 117-122.

Figure 1. SPIE-IA procedure.

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Step 1. Immunological Capture. Incubation was for 1 h at 22°C of 100 µL of E2 standard or human serum E2 standard andhuman serum samples, the latter two being diluted in EIA buffercontaining mesterolone (500 ng/mL).

Step 2. Covalent Immobilization. After washing, 100 µL ofphosphate buffer 0.1 M pH 7.4 was added followed by irradiationat 254 nm (10 J/cm2, 45 min/plate).

Step 3. Epitope Release. Next was the addition of 200 µL of 1 NNaOH for a 2-min reaction.

Step 4. Visualization. After washing, 100 µL of anti-E2 mAblabeled with acetylcholinesterase (8 EU/mL) diluted in EIA bufferwas added, followed by incubation for 3 h at 4 °C. After washing,200 µL of Ellman’s reagent20 was added and absorbance at 414nm after 30 min of enzymatic reaction was determined. The photo-SPIE-IA signal was expressed in terms of absorbance units (AU).

Competitive EIA Procedure. For the specificity study,competitive EIA was performed in EIA buffer as describedelsewhere.19 Briefly, 96-well microtiter plates were coated withaffinity-purified polyclonal goat anti-mouse IgG antibodies (Im-munotech). To each well were added 50 µL of E2 or other steroids,50 µL of E2-AChE conjugate (2 EU/mL), and 50 µL of purifiedanti-E2 mAb (10G6D6) diluted to 10 ng/mL in EIA buffer. Afteran 18-h incubation at 4 °C, the wells were washed and 200 µL ofEllman’s reagent was dispensed into each well. The absorbanceat 414 nm was measured after 30 min of enzymatic reaction.

Mouse IgG Detection. To localize mouse IgGs in molecularsieve chromatography fractions (see below), 100 µL of eachfraction and 100 µL of EIA buffer were incubated in 96-wellmicrotiter plates coated with affinity-purified goat anti-mouse IgGantibodies as described above. After 18 h of incubation at 4 °C,the wells were washed and 200 µL of goat anti-mouse IgGantibodies labeled with AChE (2 UE/mL)19 was added for 3 h ofincubation at 22 °C. After washing, enzyme activity was measuredas described in the Experimental Section after 1 h of reaction.

Calculation. The results are expressed in terms of AU at 414nm as a function of E2 concentration. Nonspecific binding (NSB)was determined at zero concentration of standard (buffer or E2-free serum). The lower limit of detection (LLD) was taken as theconcentration of E2 serum standard inducing a significant increasein NSB (3 standard deviations). The precision profile of thestandard curve was determined by performing all measurementseight times and was expressed in terms of coefficient of variation(% CV). Unknown concentrations were calculated from a standardcurve using Immunofit Software (Beckman). All measurementsof standards and samples were done in duplicate. For competitiveEIA experiments, the results are expressed in terms of B/B0 ×100 as a function of the logarithm of the steroid concentration.

Specificity Measurements. Specificity was studied by estab-lishing a standard curve for each steroid, and the results wereexpressed in terms of percentage cross-reactivity (% CR) arbitrarilydefined as the ratio of the concentration of E2 (pmoL/mL) versusthe steroid concentration producing an absorbance of 0.5 unit forphoto-SPIE-IA or a 50% decrease in B0 for competitive EIA.

Correlation Study. The validity of photo-SPIE-IA was testedby comparison of the E2 levels in 40 human sera when assayedwith two different commercial immunoassays from bioMerieux:a 125I-competitive RIA (CoatRIA 2) and an enzyme-linked fluores-

cent immunoassay performed in an automated instrument (Vidas).These two assays use polyclonal anti-E2 antibodies.

Molecular Sieve Chromatography Experiments. To studythe effects of UV irradiation on E2-antibody complexes coatedon the solid phase and to demonstrate the covalent binding of E2to the coated mAbs after the irradiation and release steps, variousincubation mixtures containing [3H]E2 were chromatographed.These experiments were performed on an AcA44 (BioSepra)column (40 × 1 cm) allowing fractionation in the 130-10 000range with EIA buffer as eluting solvent. Fractions (1 mL) werecollected and analyzed both by radioactivity counting and anti-mouse IgG immunoreactivity measurements using goat polyclonalanti-mouse IgG antibodies as capture and tracer antibodies. Twosets of experiments were conducted. In the first, after incubationof 100 pg of [3H]E2 (6000 cpm), the final supernatants of eightwells, either after irradiation or after irradiation and epitoperelease, were pooled and chromatographed. In the second, 50 µLof a mixture of [3H]E2 (500 pg) and anti-E2 mAb (60 µg) dilutedin coating and saturating buffers were incubated in uncoatedmicroplates, irradiated, and treated with 1 N NaOH beforechromatography.

RESULTS AND DISCUSSIONStudies of Experimental Conditions. Kinetics of Irradiation.

After immunological capture of an E2 (50 pg), we compared thesignal finally recovered after increasing the irradiation time at 254,312, and 365 nm (Figure 2). For each of the three irradiationwavelengths, a signal was obtained that tended to increase fasterat 254 nm than at 312 nm and rose quite slowly at 365 nm. Thesecurves never reached a plateau as expected for the saturation levelcorresponding to maximal cross-linking of the hapten. An inflec-tion point was noted close to 45 min of irradiation (10 J/cm2) at254 nm and to 160 min (40 J/cm2) at 312 nm but was not reachedafter 540 min (160 J/cm2) at 365 nm. This maximum was followedby a decrease in the signal. Interestingly, like the increase seenin the first part of the curve, this decrease was faster at 254 nmand slower at 312 nm. However, the maximal signal obtained wasequivalent for 254 nm (10 J/cm2) and for 312 nm (40 J/cm2).These data suggest that at least two contrasting events occur

Figure 2. Effect of irradiation time on the photo-SPIE-IA signal: (9)254, (0) 312, (b) 365 nm. NSB was less than 0.07 AU for allmeasurements. Inset: Binding capacity [3H]E2 (500 pg/mL) topreirradiated monoclonal anti-E2 coated solid phase at 254 nm afterdifferent irradiation times.

1004 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999

during irradiation at 254 and 312 nm. One corresponds to anincrease in the cross-linking of E2 to the solid phase and the otherto a negative effect of irradiation leading to a decreasing signalproportional to the irradiation time. This latter event may involveat least two phenomena: gradual destruction of the E2 immu-noreactivity detected by the tracer antibody and/or progressiveloss of immunoreactive material linked to the solid phase duringUV treatment. We further investigated these hypotheses.

Effects of UV Irradiation on Coated Anti-E2 mAb and E2. Weinvestigated the effect of the UV treatment on the binding site ofthe anti-E2 mAb. As shown in the inset of Figure 2, preirradiationof the coated mAb lowered the binding capacity as measured bythe capture of [3H]E2, which appears to be proportional to theirradiation time. When the coated mAb was preirradiated at 254nm (10 J/cm2) before standard SPIE-IA, the signal was equivalentto the NSB, suggesting total loss of the binding capacity of mAb.UV irradiation therefore appears to denature the binding site ofthe mAb. Preirradiation of E2 under the same conditions (or at40 J/cm2 for 312 and 365 nm) yielded the same observation. Allthese experiments point to the great sensitivity of the E2 epitopeto UV irradiation.

Individually, therefore, E2 and anti-E2 mAb are sensitive toUV-induced denaturation. However, the paratope-epitope com-plex formed after immunological capture seems to afford protec-tion against this UV-induced damage. In light of these experi-ments, we introduced a new concept of “epitope-paratope mutualprotection”, allowing the covalent linking of the hapten to the mAbwithout total denaturation of the epitope. This could account forthe initial part of the curves in Figure 2. However, the secondpart of the same curves suggests either that this protectiondecreases at more intense UV irradiation or that it correspondsto a gradual scouring of the protein solid phase. The latterhypothesis is most likely since analysis of mouse IgGs in thesupernatant of wells after various periods of irradiation at 254 nm(see Experimental Section) reveals marked similarity between thephoto-SPIE-IA signal and the mouse IgG immunoreactivity, withan inflection point close to 45 min of irradiation followed by adecrease in the signal due to gradual destruction of mouse IgGimmunoreactivity (data not shown).

Dose-Response Curves as a Function of UV Wavelength. Whenthe standards were incubated in EIA buffer and radiant exposureof 10 J/cm2 (45 min of irradiation) was used, we observed twotypical increasing dose-response curves at 254 and 312 nm(Figure 3), while the 365-nm curve was close to the NSB. Thesecurves correspond to classical immunometric assay measure-ments, suggesting that the direct UV irradiation induces efficientcovalent cross-linking, thereby allowing SPIE-IA to be performedas described for other haptens. An LLD close to 10 pg/mL wascalculated for the standard curve obtained after irradiation at 254nm (10 J/cm2). Irradiation at 312 nm (40 J/ cm2) or laserirradiation (248 nm, 40 mJ/well, 6 s of irradiation per well) usinga KrF excimer laser from Lambda Physik (Gottingen, Germany)gave standard curves superimposable on the curve obtained at254 nm and 10 J/cm2 (data not shown). Since no increase insensitivity was obtained using these protocols, 254-nm irradiationwas chosen due to the longer irradiation time at 312 nm anddifficulties in routinely performing the laser experiments.

Similar results were obtained with the three other anti-E2mAbs, with 254-nm irradiation at 10 J/cm2 always giving the bestresults (data not shown). Interestingly, the final photo-SPIE-IAresults paralleled quite closely the sensitivities obtained afterperforming a competitive EIA with these four mAbs (10G6D6,3F2B7, 5H2A10, 17E12E5). When the standards were incubatedin EIA buffer and exposed to 10 J/cm2 (for 45 min), thesensitivities expressed in terms of LLD for SPIE-IA were 10, 12,42, and 51 pg/mL, respectively, and expressed in terms of B/B0

50% for competitive EIAs were 60, 200, 500, and 550 pg/mL,respectively.

Selection of Epitope Release Agent. We previously observedduring development of SPIE-IA for various molecules that theoptimal agent for this step may vary according to the hapten andantibody. Ideally, this agent must concomitantly ensure thedissociation of the epitope-paratope complex and the denaturationof the antibody binding site to avoid any immunological recapture.We tested acids (1 N HCl, 10% formic acid), a base (1 N NaOH),organic solvents (methanol, ethanol, 1-propanol), a chaotropicreagent (8 N urea), and a detergent (10% SDS) and estimated theirefficiency: (i) by measuring the [3H]E2 released (dissociationeffect) from the [3H]E2-mAb complex; (ii) by quantifying thebinding capacity of coated anti-E2 mAb pretreated by these agents(denaturing effect). We also compared the photo-SPIE-IA signalobtained for the same concentration of E2. NaOH and formic acidwere the best dissociating agents, NaOH, HCl, and formic acidwere the most efficient denaturing agents, and NaOH andmethanol gave the best photo-SPIE-IA signal (results not shown).We therefore selected 1 N NaOH as the optimal epitope releaseagent.

Effects of the Different Photo-SPIE-IA Steps. As previouslydescribed during the development of SPIE-IAs for various mol-ecules, the two steps of the procedure (i.e., covalent immobilizationand epitope release) were strictly necessary. If they are eliminated,signal production is abolished as efficiently as if tracer additionis omitted. This confirms that the UV irradiation acts like thebifunctional chemical agents in other SPIE-IAs, by covalentlylinking the hapten to the solid-phase coated mAb.

Yield of Photo-Cross-Linking. Using [3H]E2 (50 pg), we analyzedE2 immobilization after irradiation (10 J/cm2, 254 nm) and epitoperelease and found approximately 60 and 25%, respectively, bound

Figure 3. Dose-response curves at various UV wavelengths: (9)254, (0) 312, and ([) 365 nm, (+) without irradiation.

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radioactivity with reference to the immunological capture step.The radioactive materials released in the supernatant after eitherUV irradiation (Figure 4A) or irradiation and 1 N NaOH treatment(Figure 4B) were analyzed by molecular sieve chromatography.Low-molecular-weight radioactive material was released from thesolid phase in the total volume, whereas a weak anti-mouseimmunoreactive peak not associated with the radioactivity wasdetected (Figure 4A). After NaOH treatment (Figure 4B), tworadioactive peaks were eluted, one near the void volume and theother close to the total volume. Anti-mouse IgG immunoreactivityanalysis of these fractions revealed a broad peak between the voidand total volume. This broad peak was not observed when theUV irradiation step was omitted (data not shown). These resultsshow that the NaOH treatment removes some of the moleculescoated onto the solid phase. Radioactivity analysis demonstratesthe simultaneous release of IgG-E2 conjugates and of a low-molecular-weight substance which could correspond either to freeE2 or to E2 covalently linked to degraded mAb fragments. When

[3H]E2 was incubated with anti-E2 mAb in buffer as described inExperimental Section and then irradiated and treated with NaOH,the chromatogram (Figure 4C) revealed the elution of threeradioactive peaks and a single immunoreactive material. The twoweak radioactive peaks could be attributed to native or modifiedfree E2 and the other to protein-E2 conjugate. The highradioactive peak that was coeluted with anti-mouse IgG immu-noreactivity in the void volume could be attributed to E2-monoclonal anti-E2 conjugate. As a confirmation, the bulk of theradioactivity (75-100%) was immunoprecipitated with anti-mouseIgG antibodies after addition of poly(ethylene glycol)23 (resultsnot shown). These results show that (1) E2 is linked to coatedIgG molecules and not to the proteins (serum albumin and caseinin I-Block buffer) used during the procedure, (2) some of the E2molecules could not be covalently linked to the capture antibody,and (3) coated anti-E2 mAbs are sensitive to UV irradiation andsome of them were photolyzed.

Sensitivity and Precision. The serum standard curve for E2measurement indicated an LLD close to 38 pg/mL. Assayprecision was good, with a CV less than 10% in the 40-3000 pg/mL range.

Specificity. Results of cross-reactivity studies for photo-SPIE-IA and competitive EIA are presented in Figure 5. Photo-SPIE-IAis as specific as the corresponding competitive EIA using the samemAb. The epitope recognized by the mAb appears to be locatedroughly opposite the phenolic ring. The assay appears not to sufferfrom interference by related steroids potentially present inbiological samples.

Correlation Study. E2 was assayed in 40 human sera bycomparison with the CoatRIA kit and the Vidas automat. The threeassays correlated well for samples (36 out of 40) measured in theconcentration range of the standard curve (40-3000 pg/mL).Correct quantification for the four other samples required dilutionin E2-free serum for a good fit. Linear regression analysis gavethe following equations: photo-SPIE-IA ) 1.04CoatRIA - 0.08, r2

) 0.95 and photo-SPIE-IA ) 1.00Vidas - 0.29, r2 ) 0.93.

CONCLUSIONSWe have demonstrated that 17â-estradiol can be cross-linked

to a coated anti-E2 mAb by direct UV photoreaction. Althoughboth the hapten and the paratope of the mAb appear quitesensitive to UV irradiation, which denatures them, the immunore-activity of E2 bound to the paratope seems at least partly preservedas revealed by the final recognition and binding of the tracerantibody. However, the results presented here raise several keyquestions: (1) Which site in the E2 structure is involved in cross-linking? It is currently agreed that the photo-cross-linkingsproceed through the generation of free radicals from a chemicalgroup (donor) followed by fast reaction with its environment(acceptor). The consequence is a modification of ligand structurewhich in our case does not greatly alter immunoreactivity towardthe tracer antibody. Using synthetic E2 analogues as 6-CMO-E2,6-keto-E2, 2-bromo-E2, 2-hydroxy-E2, 4-bromo-E2, 4-hydroxy-E2,and 3-hemisuccinate-E2, study of the specificity shows thatpositions 2, 3, 4, and 6 in the E2 structure are not located in theepitope, these molecules being well recognized by the mAb (datanot shown). As a consequence, we hypothesize that the area of

(23) Hartmann, D. J.; Grassi, J.; Ville, G. J. Immunoassay 1993, 14, 241-266.

Figure 4. Chromatograms of radioactive (O) and anti-mouseimmunoreactive (b) materials present either in supernatants afterirradiation followed by epitope release or in the mixture of incubationmedium of [3H]E2 and anti-E2 monoclonal antibody after irradiationand NaOH treatment (C): v0, void volume; a.p. alkaline phosphatase;perox., peroxidase; vt, total volume.

1006 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999

cross-linking to the E2 molecule is close to the hydroxyl group.Moreover, E2 possesses a phenolic ring known to be easilyconverted to highly reactive phenoxyl radicals24 by UV irradiation.In terms of the location of the site of cross-linking to the antibody,numerous amino acid side chains such as tryptophan25 or glutamicacid26,27 could be involved in the photoreaction, as reported forthe study of various receptors or binding proteins by direct UVphotoaffinity labeling. (2) How can the low yield of E2 cross-linkingto the coated monoclonal antibody be explained? It is well-knownthat 254-nm irradiation inactivates enzymes28 through photolysisof disulfide bridges and aromatic residues. Few papers havereported the effects of direct UV exposure of antibodies, such asthe loss of binding capacity29 and a tendency to aggregate.30 Amore recent study of direct photoactivation of a monoclonalantibody for technetium-99m labeling31 reported an 85% yield ofintact whole mAb after an irradiation procedure similar to thatdescribed here. Our chromatographic results recorded whenhapten-antibody complex was irradiated and treated by NaOHin solution (Figure 4C) are in agreement with these previous

findings. Nevertheless, under our experimental conditions usingcoated antibody and the same process, we demonstrate thatfragmentation of IgG molecules may occur. (3) What is therelationship between the irradiation wavelength and photo-SPIE-IA? We analyzed the possible relationship between the irradiationwavelength and the efficiency of cross-linking in SPIE-IA experi-ments using a monochromator delivering weak energy andallowing irradiation every 10 nm in the 230-380-nm range.Preliminary results (data not shown) reveal two peaks of the photo-SPIE-IA signal at 290 and 240-250 nm. We are now developing aUV-generating device able to deliver UV light at various wave-lengths with high energy in the hope of increasing the cross-linking yield. (4) Could this procedure be extended to other hapten-antibody systems? Since positive results have been obtained forthe E2 molecule with four different mAbs, it can be reasonablyassumed that this irradiation procedure could be extended to othermolecules, although our results are as yet preliminary. We willapply this photo-SPIE-IA to other steroid molecules such asprogesterone and cortisol, in the hope of acquiring similar results.Moreover, we have demonstrated the application of this procedureto angiotensin II (1048) with an LLD close to 5 pg/mL when thestandards were incubated in EIA buffer and exposed to 10 J/cm2

(for 45 min) at 365 nm (manuscript in preparation). Photo-SPIE-IA of histamine (111) was also successfully under investigationwhen the histamine was prederivatized by photoreactive probeprior to assay (work in progress).

With this new technology and commercial UV sources andvarious monoclonal anti-hapten antibodies, we have successfully

(24) Creed, D. Photochem. Photobiol. 1984, 39, 563-575.(25) Grenot, C.; Blachere, T.; Rolland de Ravel, M.; Mappus, E.; Cuilleron, C. Y.

Biochemistry 1994, 33, 8969-8981.(26) Carroll, S. F.; Collier, R. J. Methods Enzymol. 1994, 235, 631-639.(27) Cockle, S. A. FEBS Lett. 1989, 249, 329-332.(28) Luse, R. A.; McLaren, A. D. Photochem. Photobiol. 1963, 2, 343-360.(29) Tarkchanova, I.; A.; Schanin, S. S.; Kulberg, A. Y. Biochim. Biophys. Acta

1969, 175, 463-465.(30) Wickens, D. G.; Norden, A. G.; Lunec, J.; Dormandy, T. L. Biochim. Biophys.

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Figure 5. Comparison of cross-reactivity (%) observed with a competitive enzyme immunoassay and photo-SPIE-IA tested using varioussteroids.

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developed a sensitive photo-SPIE-IA. It is worth noting that twodifferent 17â-estradiol analogues, possessing in position 3 or 6 analiphatic short chain ending with an amine group, are very effectivein SPIE-IA with glutaraldehyde as cross-linking reagent and thesame monoclonal anti-E2 antibody (results not shown). Finallywe hope to optimize photo-SPIE-IA of 17â-estradiol in terms ofcross-linking efficiency by investigating the use of a moreappropriate UV-delivery system and, in terms of sensitivity, bygenerating monoclonal anti-E2 antibody tracer with a high affinityfor photolinked E2. Other irradiating sources such as ionizingirradiation (γ rays) have yielded very promising results and willalso be further investigated.

ACKNOWLEDGMENTThis work was supported by grants from the Commissariat a

l′Energie Atomique and bioMerieux (France). We thank bioMerieuxand the Agence Nationale de la Recherche Technologique for afellowship awarded to L.B. We thank Dr. Christophe Moulin(DCC/DPE/SPCP) of the Commissariat a l’Energie Atomique forhis help in preliminary laser experiments.

Received for review August 4, 1998. Accepted December3, 1998.

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