High-Affinity Uranyl-Specific Antibodies Suitable for Cellular Imaging

Chem. Res. Toxicol. 2008, 21, 349–357

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High-Affinity Uranyl-Specific Antibodies Suitable for Cellular Imaging Laetitia Reisser-Rubrecht,† Caroline Torne-Celer, Wendy Rénier, Olivier Averseng, Sophie Plantevin, Eric Quéméneur, Laurent Bellanger, and Claude Vidaud* CEA Valrhô, DSV/IBEB/SerVice de Biochimie et de Toxicologie Nucléaire, BP 17171, F-30207 Bagnols sur Cèze, France ReceiVed June 14, 2007

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO22+-DCP-BSA (DCP, 1,10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO22+-DCP-casein, we selected two highly specific mAbs against uranyl-DCP (KD 10–100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO22+, Fe3+, Zn2+, Cu2+, and Ca2+) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO22+ equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immunolabeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its paratopes can accommodate a wide variety of chelating groups. Introduction Uranium is a naturally abundant actinide on Earth and has been heavily used in many chemical forms in civilian and military industries. It is now widely accepted that its chemical toxicity in addition to its radioactivity should be taken into account and deserves to be further documented. Its natural occurrence in the environment and its daily ingestion via drinking water have been the subject of studies (1). Whatever its route of entry into the body, uranium reaches the blood and is partly stored in target organs such as bones and kidneys (2, 3). Uranium is mostly found in the form of hexavalent uranyl ions (UO22+) in aqueous media. It has no direct chemical equivalent, and understanding its mechanisms of toxicity relies on the identification of relevant molecular targets and accurate knowledge of the functional consequences of metal-target interactions. Interactions of such a complex metal ion with different proteins or small molecules are largely influenced by its complicated coordination chemistry (five to six possible ligands in its equatorial plane and strong electrostatics). Furthermore, the cell transport of this metal still remains to be described. Some target proteins have already been identified for uranyl ions (4, 5), but few uranyl binding sites have been well-described (6, 7). The development of high-affinitiy monoclonal antibodies (mAbs) could be an interesting approach to obtain a biologically accessible subset of metal-binding proteins and provide additional structural information. Metal ions are known to be able to induce an immune response (8), and serological assays have been developed to detect antimetal antibodies, for example, against beryllium (9). Reardan et al. (10) were the first to * To whom correspondance should be addressed. Tel: +(33)4-66-7967-62. Fax: +(33)4-66-79-19-05. E-mail: [email protected]. † Current address: FRE 3009 CNRS/Bio-Rad, Faculté de Pharmacie, 15, avenue Charles Flahault, BP 14491,34093 Montpellier Cedex 5, France.

describe mAbs raised against metal-chelate immunogens. Some authors have also developed different strategies based on designing bispecific antibodies, with one of the two specificities directed against metal complexes (11, 12). These antibodies might be used as tools for tumor imaging or therapeutic applications by acting as pretargeting agents (13–19). For such applications, metal chelates are administered once the antibodies are prelocalized at their specific target organs. Since 1992, different authors have reported mAbs directed against different metal-chelate complexes (13, 20–23), including antiuranyl antibodies (24, 25). They displayed dissociation constants in the nanomolar range and were sensitive enough to detect traces of UO22+ in the environment. The chelating agent 1,10phenanthroline-2,9-dicarboxylic acid was successfully used and looked strong enough to preserve metal-chelate complexes during immunizations. Our aim was to generate a new set of mAbs directed toward uranyl ions for further investigation of uranyl coordination chemistry and novel applications in the field of toxicology. In particular, we wondered whether it was possible to generate mAbs whose paratopes might mainly recognize the uranyl moiety in the chelate complex and eventually cross-react with different chelates from those used as an immunogen. We raised several mAbs against uranyl-bound 1,10-phenanthroline-2,9dicarboxylic acid (UO22+-DCP). We specifically focused on two of them with different isotypes, U04S (IgG2b) and U08S (IgG1), and striking biochemical features.

Experimental Procedures Reagents. All chemicals, except Alexa Fluor 488 or 647 (Molecular Probes, Invitrogen, Oregon), were from Sigma-Aldrich. Alexa Fluor 488 mouse IgG2b isotype control was from Invitrogen (Cergy Pontoise, France), and G25 chromatographic support was

10.1021/tx700215e CCC: $40.75  2008 American Chemical Society Published on Web 12/22/2007

350 Chem. Res. Toxicol., Vol. 21, No. 2, 2008

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Table 1. Characterization of Stoichiometries for Protein Conjugatesa

BSA casein

DCP/protein molar ratio

U/protein molar ratio

12.5 ( 0.5 4.5 ( 0.5

5 ( 0.5 1.5 ( 0.5

a The DCP/protein ratio was evaluated after desalting steps by DCP absorbance at 340 nm, and the uranyl /protein ratio was measured by ICP-MS. The amount of protein was evaluated by BCA assay. One representative set of protein conjugates was used in this study.

Figure 2. Interaction with free metal cations monitored by SPR competition assay. Solid-phase U04S and U08S mAbs were incubated with increasing quantities of different metal cations (uranyl acetate, FeCl3, ZnCl2, CuCl2, and CaCl2) prior to injection of UO22+-DCPcasein. Results are average values of duplicates with CV > 5%. The percentage of conjugate binding inhibition is expressed as a logarithmic function of cation concentration, relative to the uranyl inhibition signal as a reference. Figure 1. SPR analyses of conjugate binding to mAbs U04S and U08S. mAbs were immobilized on the surface of the sensor chip and protein–ligands were analyzed in real-time analysis. Signal levels were normalized for 1000 RU of each antibody and compared with a nonspecific antibody (NS), an anti-KLH mAb. Results are expressed in moles of bound conjugate. (A) Direct interaction with DCP-protein conjugates and effect of UO22+ loading on protein conjugates. (B) Protection of interaction by prior saturation of the solid phase with uranyl acetate (+). UO22+-DCP conjugate interaction is prevented when the paratopes of solid-phase immobilized mAbs are saturated with free uranyl and enhanced for the free DCP conjugate.

from GE Healthcare. TRIS-buffered saline (TBS) was composed of 20 mM TRIS-HCl and 0.15 M NaCl, pH 7.4. Tween TRISbuffered saline (TTBS) contained 0.05% Tween 20. Synthesis of 5-Isothiocyanato-1,10-phenanthroline-2,9-dicarboxylic Acid. The compound was synthesized (ERAS labo, Saint Nazaire les Eymes, France) in five steps using neocuproin hydrate as the starting material. The product was converted to 5-nitro-2,9bis(trichloromethyl)-1,10-phenanthroline and then in two steps to 5-amino-1,10-phenanthrolin-2,9-dicarboxylic acid. The amine function was transformed into an isothiocyanato derivative. 5-Thiouredoethanethiol-1,10-phenanthroline-2,9-dicarboxylic acid (DCP-thiol) was obtained in one step from the last product using aminoethanethiol chlorhydrate in pyridine as the solvent. Preparation of Immunogen and Protein Conjugates. DCPisothiocyanato and proteins were mixed in 50 mM HEPES buffer, pH 9, with gentle stirring overnight and at room temperature. Reagent/protein molar ratios were 100:1 and 20:1 for BSA (bovine serum albumin) and κ-casein, respectively. Desalting was performed by exclusion chromatography on a G25 column with 50 mM HEPES, pH 7.4, as the mobile phase. The DCP content was evaluated using the 340/280 nm absorbance ratio. The amount of protein was calculated via a BCA (bicinchoninic acid) assay.

Uranyl-DCP-protein conjugation was achieved by mixing 2 mol of uranyl acetate per mol of DCP-protein in 50 mM HEPES and sodium carbonate, pH 7.4, overnight at room temperature. After extensive dialysis against 50 mM HEPES, pH 7.4, the uranyl content was determined by ICP-MS as described earlier (26). Metal complexes were stored at -20 °C. The UO22+-HSA (human serum albumin) complex was prepared as previously detailed (26). Direct labeling of the Mabs was achieved with Alexa Fluor 488 or 647, using succinimidyl chemistry and according to supplier instructions. They were analyzed spectrophotometrically after coupling and desalting on G25. The fluorophore content was within 3–4 mol per mol of mAb. Preparation of Hybridomas and mAbs. UO22+-DCP-BSA as an immunogen was emulsified 50:50 with Freund’s adjuvant (Sigma, France). Two groups of four BALB/c mice (Charles River, France) were each immunized by intraperitoneal injections of 10 or 20 µg of immunogen. Booster injections were given at the same doses at 4 week intervals using Freund’s incomplete adjuvant (Sigma, France). Fourteen days after each boost, the animals were bled and their sera were tested by ELISA (enzyme-linked immunosorbent assay) for antibodies reacting with UO22+-DCP-casein (see below). Three days before cell fusion, an intravenous injection of the immunogen, in 0.9% saline solution, was administered. A P3X63Ag8.653 myeloma cell line (27) was fused with splenocytes from immunized mice according to standard protocols (28). Antibody-producing hybridomas were subcloned and then frozen in liquid nitrogen. mAbs were produced in vivo by intraperitoneal injection of hybridoma cell lines into BALB/c mice. mAbs were then purified from ascitis fluid by ammonium sulfate precipitation (70% saturation) and affinity chromatography on protein A-sepharose (Biosepra, France). Antibody Selection and Characterization. Indirect ELISA screening was used for counterselecting anti-BSA hybridomas. UO22+-DCP-casein or DCP-casein (10 µg/mL in 50 mM HEPES

Anti-Uranyl Antibodies and Cellular Imaging

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Figure 3. SPR equilibrium analysis. Duplicate injections of UO22+-DCP-BSA on two U04S and U08S surface capacities (A, 400 RU; and B, 160 RU). UO22+-DCP-BSA concentrations (nM) are indicated on the curves. To avoid mass transfer, the flow rate was fixed at 30 µL/min. The protein solutions were injected for 90 s, and depending on the antibodies, conjugates were allowed to dissociate for 300 or 600 s after the association phase. Association and dissociation constant values were calculated using the Biaevaluation 3.0 software (Biacore, GE Healthcare).

pH 8) was absorbed onto 96-well microtiter plates (ImmunoPlate Maxisorp, Nunc, Denmark) for 3 h at 4 °C. After washing, the coated plates were saturated overnight at 4 °C using 100 µL of fish gelatin at 5 g/L in the same buffer. Immunsera (10-1 to 10-8 dilution in 50 mM HEPES, pH 7.2, 0.15 M NaCl, and 3 g/L BSA), culture supernatants, or purified antibodies were incubated (100 µL/ well) for 3 h at room temperature with agitation. The revelation system was the classical HRP-conjugated goat antimouse IgG (1:50 000) using tetramethyl-benzidine as a substrate. Surface Plasmon Resonance (SPR) Analyses. The Biacore 2000 (Biacore, GE Healthcare) was used to evaluate specific antibody subclasses, antibody-conjugate interactions, and affinity parameters. Antimouse antibodies anti-Fc (R-mouse Immunoglobulins, Biacore, GE Healthcare) were covalently immobilized on a CM5 sensor chip according to the manufacturer’s recommendations in the standard EDC/NHS (N-[3-dimethylaminopropyl]-N′-ethylcarbodiimide/N-hydroxysuccinimide) and carbohydrate coupling chemistry. Ethanolamine was used to block any remaining free ester groups. Working buffers and general conditions were 50 mM HEPES, 0.15 M NaCl, pH 7.2, and 0.005% (v/v) Tween 20, at 5 µL min-1. The sensor chip was regenerated using 10 mM NaOH, 0.15 M NaCl, and then 0.1 M HCl (10 µL) before any new sample analysis. Mouse Ab subclass kit (Biacore) was used to determine isotype subclasses. Experiments were performed according the manufacturer’s instructions. Cell Imaging Experiments. HK2 proximal tubular cells (ATCC, CRL-2130) were grown in serum-free keratinocyte medium (Invitrogen, France) supplemented with 5 ng/mL recombinant epidermal growth factor, 50 ng/mL bovine pituitary extract, and 50 µg/ mL to 50 U/mL penicillin-streptomycin (Gibco). The cells were maintained at 37 °C in a 5% CO2/air incubator. The uranium solution used for exposure was prepared by diluting the uranyl acetate stock solution in 0.1 M NaHCO3, pH 8, to obtain a 10 mM UO22+ solution. A second dilution was performed in serum-free culture medium to obtain a final concentration of 0.5 mM UO22+ in the cell samples. For flow cytometry experiments, U04S was directly labeled with AlexaFluor 488 using succinimidyl ester chemistry and compared with an AlexaFluor488 mouse IgG2b (Invitrogen, Cergy Pontoise, France) as isotype controls. Samples of 105 HK2 cells were incubated for 60 min in 500 µL of keratinocyte medium containing uranium. After uranium exposure, cells were centrifuged for 10

min at 1100 rpm and fixed directly using paraformaldehyde (4%, 20 min at 4 °C). The cells were rapidly washed in PBS (phosphate buffer saline) and centrifuged. The AlexaFluor 488-labeled antibodies, diluted in PBS supplemented with 1% normal mouse serum, were added to the cells to reach a final concentration of 5 µg/mL under 100 µL. The cells were incubated with the mAbs for 45 min at 4 °C under agitation and finally diluted with 900 µL of PBS. The fluorescence intensity of each cell was recorded on a FACSCalibur flow cytometer (Beckton Dickinson, France) after excitation at λex 488 nm and collection at λem 530 ( 15 nm. Series of 10000 events were counted for each condition, and data were processed using CellQuest software (Beckton Dickinson). Confocal microscopy experiments were run on a Nikon Eclipse TE 2000-E microscope, equipped with an X60 oil objective lens. The control IgG2b used in microscopy experiments was a proprietary antibody (HCD15S). U04S and the IgG2b control were directly labeled with AlexaFluor 647 using succinimidyl ester chemistry. HK2 cells (200000 cells per chamber) were grown on LabTek chambers (Nunc, Denmark). After similar and previously described intoxication, the cells were rinsed with PBS. The cells were then fixed and permeabilized with cytofix/cytoperm solution (BD Biosciences, France) for 20 min at 4 °C, rinsed with Perm/ Wash Buffer, and then contacted with 10 µg/mL of labeled-mAb in Perm/Wash Buffer for 45 min at 4 °C. Chromatin staining was achieved in 1 µg/mL Hoechst 33342 dye (Molecular Probes, France) after two successive washes. Slides were mounted using glycerol 90% in PBS. Excitation wavelengths were 408 and 637 nm. Emission pictures were collected using 515/30 BP for Hoechst and 650 LP for the labeled antibodies. Intensity data were acquired and processed using Lucia (4.2) and EZ-C1 (3.30) Nikon softwares.

Results Although many mAbs against metal ion chelates have already been reported, it is still a difficult task to generate antibodies with appropriate specificity. Our need was to select immunochemical tools suitable for toxicological assays, that is, able to recognize uranyl in a large variety of complexed forms. Special care was then taken in the immunization and selection procedures, particularly in terms of reagent characterization. Protein conjugates were critical reagents for the success of the experiments. We carefully evaluated the DCP ratio per



Table 2. Kinetic and Thermodynamic Parameters of U04S and U08S mAbsa antibody U04S a U04S b U08S a U08S b

Rmax (RU)

ka (M-1 s-1)

30.0 4 20.0 6.0

3.4 × 10 1.8 × 106 5.4 × 105 1.20 × 106 5

kd (s-1) (2.2 × 10 -7) 1.4 × 10 -4 2.9 × 10 -5 2.0 × 10 -4

KD (M) -13

(6.4 × 10 ) 7.6 × 10-11 5.3 × 10-11 1.8 × 10-10

χ2

KD (M)

0.469 0.183 0.141 0.254

0.8 × 10-10 1.8 × 10-10

a Binding reactions with two active surface capacities: a, #400 RU; and b, #160 RU. Rmax represents the signal equivalent to saturation of the active surface by the UO22+-DCP-BSA conjugate. Results in italics are outside the specification range of Biacore 2000. The order of magnitude of KD values was deduced from both experiments.

protein and uranyl loads for UO22+-DCP-proteins using UV absorbance and ICP-MS (Table 1). Whatever the DCP content in protein, the final uranyl/DCP ratios were within the same range, that is, ∼one uranyl for three DCPs after thorough desalting steps. This partial occupancy of DCP sites, however, proved to be sufficient for generating a specific immune response in mice. Eight mice were immunized by intraperitoneal injection of UO22+-DCP-BSA conjugates and were used to obtain mAbs. To select antibodies raised against either the metal-chelate or the free chelate, the hybridoma selection procedure was stringent. We chose casein conjugates for mAb selection to avoid interference from bovine serum albumin. ELISA screening (data not shown) of 3072 culture supernatants from two fusions led to only 0.5% of them being inhibited either by UO22+-DCPcasein or DCP-casein, that is, six and two clones, respectively. To confirm that they were monoclonal, the hybridomas were subcloned twice by limiting dilutions, producing eight selected clones that could bind either the UO22+-DCP-casein or the DCP-casein complexes. Three of them were selected for their good growth characteristics. U04S 01E8 (U04S) and U08S 01C10 (U08S) could bind UO22+-DCP-casein more specifically, while another one, PHE03S 07D9 (PHE03S), bound the DCP-casein complex but the UO22+-DCP-casein very poorly. PHE03S was preserved for further studies while the other two clones, U04S and U08S, were produced via ascitis fluids and purified on a protein A column. Isotypes of the two antiuranyl mAbs were determined by SPR analysis; U04S is an IgG2b while U08S is an IgG1 (experimental data not shown). mAbs U04S and U08S Are Specific to Uranyl Ions. The specificity study of various metal cations was achieved mostly by SPR analyses. Antimouse antibodies were covalently fixed by amine coupling chemistry on the surface of CM5 sensor chips. Subsequent binding of the two specific mAbs on these surfaces led to “active” surfaces. After each cycle and sensor chip regeneration, the active surfaces were regenerated in a strictly identical way. The sensor chip was then reloaded with the specific mAb. UO22+-DCP-conjugates, with either BSA or casein, were then tested for their interaction with immobilized mAbs U04S or U08S. Binding levels were normalized to allow direct comparison of the specific mAb and were average results of duplicates or triplicates. As presented in Figure 1A, we confirmed that U04S and U08S antibodies bound both UO22+-DCPconjugates with signal levels depending on the protein type, that is, BSA or casein. The hypothesis of partial BSA recognition was eliminated because the antibodies were only counterselected against casein conjugates. Therefore, these differences were attributed to the molecular weights of the two proteins giving different refractive indices on the surfaces. A slight difference in the ability to bind solid-phase protein conjugates was also observed in favor of U04S. On the other hand, DCP-conjugate recognition was low, in both ELISA and SPR, highlighting the importance of the uranyl cation for recognition. This specificity for the metal ion was then explored. It is not technically possible to quantify the direct binding of small

Figure 4. Immunodetection of uranyl in dot-blotted protein samples. Detection of decreasing quantities of dotted UO22+-DCP-BSA, UO22+-DCP-casein, and UO22+-HSA complexes by U04S, U08S mAbs, and their related isotypic controls. Membranes were prepared by spotting 1 µL of two dilutions of proteins in TBS buffer, #14 × 10-5 M (HSA reference), #14 × 10-5 M (UO22+-HSA samples), #2 × 10-7 M (UO22+-DCP-BSA), and 6 × 10-7 M (UO22+-DCPcasein) on pregridded nitrocellulose membranes. After nonspecific sites were saturated for 2 h with 0.05% fish gelatin in TTBS, the blots were probed with 10 µg/mL mAbs in gelatin-TTBS buffer for 1 h and 30 min and revealed by HRP-conjugated antimouse antibody (1/50000) in gelatin-TTBS buffer and BM blue substrate (Roche Applied Science, France) according to standard protocols. Relative uranyl concentrations in UO22+-protein samples are indicated in the figure.

ligands with Biacore 2000, because the refractive index variation is too small to be measured reliably. Uranyl cation specificity was then confirmed by competition between various metal cations and protein conjugates. As shown in Figure 1, U04S and U08S both induced a significant signal of interaction with UO22+-DCP-casein. We performed a first injection of uranyl acetate solution prior to the different casein conjugates and measured their binding levels (Figure 1B). Uranyl acetate antagonized UO22+-DCP-casein binding to both U04S and U08S antibodies. Once again, a slight but significant difference in favor of U04S was observed. Nevertheless, UO22+-DCPcasein binding was not totally abolishedby uranyl acetate, suggesting some involvement of DCP groups in the epitope. Interestingly, initial saturation of the paratopes with uranyl acetate allowed efficient DCP-casein binding, restoring a correct signal. These results proved clearly that uranyl was essential for U04S and U08S binding to DCP-conjugates. To confirm uranyl selectivity, we then tested UO22+-DCPcasein binding inhibition by other different metals such as iron, zinc, copper, and calcium. They are all Lewis acid metals like uranyl and are relatively abundant in living organisms. The ranges of concentration of these metals were limited by their solubility in the running buffer. The extent of competition was assessed by quantifying residual UO22+-DCP-conjugate binding after prior metal contact with the two mAbs. The reported signals are relative signals, using uranyl species competition as a reference (Figure 2). As observed previously, conjugate binding was not totally inhibited for either or both antibodies even at the highest concentration of uranyl acetate, but it was significantly decreased after uranyl acetate contact for both of them, particularly for U04S, which was assumed to have the



Figure 5. Flow cytometry analysis of UO2 2+-treated cells. HK2 cells were incubated with (+) or without (-) 0.5 mM uranyl. After suitable fixation and washing steps, the cells were directly stained with AlexaFluor 488 labeled U04S or irrelevant mAb as an isotype control (Invitrogen). A series of 10000 events were counted. The fluorescence intensity of each cell was recorded after excitation at λex 488 nm and collection at λem 530 ( 15 nm. The cell morphology was plotted as SSC (cell granularity) as a function of FSC (cell size). A homogeneous cell population of regular morphology was gated from nonintoxicated control cells and was reported to be identical in the different studies. The fluorescence related to each population was presented as histograms. The percentage of gated events and the corresponding median values are indicated in the panels.

highest specificity for uranyl acetate. Adding the other metals had very little effect on conjugate binding, even with concentrations at least 10 times higher than uranyl acetate. Uranyl specificity was demonstrated for both antibodies, and the experiments proved that this cation was essential in the recognition of DCP-proteins. mAbs U04S and U08S Display High Affinity for UO22+DCP Conjugates. The goal of this study was to measure dissociation parameters between the antibodies and the different UO22+-DCP-BSA conjugates. Unfortunately, under common experimental conditions, the purified antibodies, U04S and U08S, bound so strongly to these conjugates that it was impossible to measure dissociation phenomena correctly. To discriminate between “avidity” and very high affinities, we performed our experiments under two low active surface capacities (160–400 RUsresonance unitssfor the larger one and 25–35 RU for the smaller one) and at very low conjugate concentrations (29, 30). Under such low active surface capacity conditions, specific binding signals were subsequently very low. BSA conjugates could only be injected because the refractive index modifications were large enough to ensure a correct signal/ noise ratio and thus produce reliable results, which was not the case for the casein conjugates. Each concentration of BSA conjugates was tested in duplicate. The association phases were monitored for 90 s, which was long enough to observe the curvature of the binding responses. Five or 10 min of dissociation time was necessary to calculate the dissociation rate kd reliably. Figure 3 shows an experiment with duplicate injections of 3.5 to 60 nM UO22+-DCP-BSA conjugates over the two different surfaces. Rmax responses of 25–35 RU and 5–10 RU were measured, respectively. Blank injections (aliquots of

running buffer alone) were alternated with the conjugate injections. They showed flat responses, indicating an efficient regeneration protocol and the absence of baseline drifting. Experimental reproducibility was confirmed by good duplicate results even at lower surface capacities. Kinetic fits were used to determine ka and kd, and the KD was calculated from kd/ka using Biaevaluation software. Whatever the surfaces, the different experiments matched 1:1 kinetic interaction models (with global Rmax) and led to high affinities (Table 2). In the case of U04S at the highest surface capacity, kd was outside the range of Biacore 2000 specifications, but the duplicates were reliable enough to estimate the KD, which was the least favorable of the values obtained by both experiments with the two surface capacities. These preliminary characterizations, in particular the results of high affinity against metal-chelate, were essential because U04S and U08S mAbs were intended to be used to recognize biological complexes of uranyl. Different experiments were then planned to evaluate uranyl detection in proteins. mAbs U04S and U08S as Tools for Detecting Uranyl Protein Complexes. Feasability of Detection by Dot Blots Experiments. Previous results showed that uranyl acetate could displace the equilibrium between mAbs and UO22+-DCPproteins. Direct uranyl-protein detection was investigated, first with chelate-free UO22+-HSA complex. The protein was prepared as described previously (26). Under such conditions, a metal content of 0.25 mol of uranyl per mol of protein was determined by ICP-MS after G25 desalting. The metal-binding sites may be solvent exposed and accessible to competing groups, leading to a final low-binding level. Protein quantities in the dot blot experiments were chosen taking into account



Figure 6. Fluorescence microscopy. Cells were cultivated with 0.5 mM uranyl (+) or without uranyl (-) as a control culture. Different incubations with AlexaFluor 647-labeled antibodies were performed after cell fixation and permeabilization with Perm/Wash Buffer solution (BD Biosciences, France). Hoechst 33342 dye was used for chromatin staining. -A, phase contrast; -B, Hoechst 33342 staining; -C, Alexa Fluor 647 fluorescence; and -D, merge.

this low uranyl over HSA ratio. As shown in Figure 4, a light marking of UO2 2+-HSA spots was observed with both mAbs U04S and U08S, whereas metal-free HSA spots showed no labeling. Uranium concentrations in these spots ranged from 1.8 to 3.5 × 10-5 M. The signal level was, as expected, significantly lower than that of the UO22+-DCP-BSA sample. Furthermore, we could not exclude that part of the uranium was removed from the DCP-free HSA by the different washing steps. Flow Cytometry Analysis of Uranium Treated Cells. We explored the usefulness of the more specific antibody U04S in detecting uranium in intoxicated cells through flow cytometry analysis. UO22+ speciation in a complex culture medium has not yet been precisely determined although theoretical speciation has been calculated using programs such as J CHESS and BASSIST (31, 32). Conditions for intoxication were adapted to reduce the time of metal exposure and optimized to limit

cell stress by multiple washing steps and centrifugations. U04S and an Alexa Fluor 488 mouse IgG2b isotype control were used to detect uranium in cell populations. Figure 5 presents the morphological characteristics of control and stained cell populations and their corresponding fluorescence histograms. Forward scatter (FSC) is a measure of cell size, and side scatter (SSC) is a measure of cell granularity or general shape. From these plots, a homogeneous population with regular morphology (∼97% of total population) was gated from normal HK2 cells, and this selected area was reported in each panel of the other experimental results. A slight fluorescence background was observed for each control populations (left-hand columns and upper line, right panel) as compared with normal HK2 fluorescence. In particular, calculation using the statistical tool of the software showed that the median values of the corresponding fluorescence histograms were slightly higher than in a normal



Figure 7. Visualization of intracellular uranyl species by confocal microscopy (stack no. 20/44): representative UO22+-treated cells stained by AlexaFluor 647-labeled U04S. -A, Hoechst 33342 staining; -B, Alexa Fluor 647 fluorescence; -C, merge; and -D, zoom on the gated area.

cell population. The cells displayed some altered morphology after intoxication (columns on right-hand side) with a shift toward broader granularity (SSC). Nevertheless, the homogeneity of gated cells was around 76% of the total population for each intoxication experiment. Some fluorescence background was observed for the IgG2b isotype control, but clearly, the fluorescence level of the gated population was significantly shifted when the intoxicated cells were stained with U04S. In particular, the median value was four times higher for U04S than for the IgG2b control, which proved the capacity of U04S to bind particular uranyl species in a more specific way. Immunodetection of Uranyl Accumulation in HK2 Cells. In the previous experiments, the fluorescence of intoxicated cell populations did not give any information about the fraction of internalized metal ions. The use of specific antimetal antibodies to confirm the location of intracellular metal would be a major achievement. However, uranium establishes many associations with salts, small molecules, macromolecules, and organelles. Such an application would require an mAb with rare features, such as those exhibited by U04S. The experimental conditions of cell-metal contact were analogous to those used in the previous experiment. Only cells grown in uraniumcontaining medium and incubated with AlexaFluor647- U04S displayed significant staining (Figure 6, bottom line). No significant labeling was observed in any of the control samples, either in intoxicated cells stained with irrelevant antibody or in nonintoxicated control cells stained with U04S antibody. The majority of the cells were stained red when U04S was used, as could be observed in Figure 7B,C and in agreement with fluorescence-activated cell sorting (FACS) results. These con-

focal field observations proved that the uranyl species were not coated onto the surface as the staining was clearly intracellular. The spatial distribution of uranyl was confirmed in the detailed cells of a zoomed area (Figure 7D). Labeling was mostly cytoplasmic, even if a few cells also exhibited little red spots in the nucleus. Staining was much more intense in some particular cytoplasmic regions, thus indicating higher uranyl concentration. The nonspecific fluorescence background of IgG2b control, previously observed in FACS experiment, was undetectable in confocal field observation. However, the quantum yield is lower for AlexaFluor 647 than for AlexaFluor 488 dye. The total uranium concentration in the intoxicated cells was measured by ICP-MS, assimilating HK2 cells to 18 µm diameter spheres. An estimate of 100–200 µg uranium per g of cell was found. This result is original and suggests that the process of uranyl uptake by HK2 cells is fairly fast, since the cells were observed after only ∼60 min of incubation with the metal solution. This last set of experiments provided evidence that a mAb can be an efficient tool for detecting and monitoring different uranyl species in intoxicated cells.

Discussion Antibodies are exquisite binding proteins, and the aim of our study was to generate and characterize specific antiuranyl antibodies, with the double objective of simultaneously having a sensitive tool to detect uranyl species in biological samples and an appropriate model for studying metal binding sites in target proteins. Different antimetal antibodies have already been described with affinities ranging from 10-8 (33) to 10-12M (34).


These high affinities are probably related to a large contact area between amino acid residues in the antibody paratope and the chelated metal ion, with significant contribution of chelating functions over the metal itself (34). In this study, we immunized mice with UO22+-DCP-BSA and selected two mAbs with the advantage of having two different isotypes. U04S and U08S exhibited strong reactivity toward UO22+-DCP-proteins as compared with metal-free DCP-proteins. SPR analysis revealed that they display high affinities, the best reaching a 10-11 M range. In a competition assay, uranyl acetate was the only cation of the different hard Lewis metals tested to be able to limit UO22+-DCP-conjugate binding significantly, demonstrating the specificity of both antibodies for this metal. Strikingly, both mAbs were able to detect uranyl-HSA in dot blot experiments but with a low binding level. HSA can slightly bind uranyl cations, and to allow additional ligation between the metal and the antibody, the coordination shell of the metal in HSA must probably be partially filled as suggested by some authors (34). Because neither antibody could bind HSA alone, it was obvious that the HSA-antibody interaction was the result of interfacial metal. The weak binding was explained by competition between the HSA and the antibodies, a labile uranyl-HSA interaction, and also the need for different washing steps in such experiments. In situ detection of essential or toxic metal ions is an important field of investigation. Here, we present a mAb as a specific probe for uranyl species, which enabled us to image some particular uranyl species in cells selectively and reach the detection limit within the range of electron microprobes (35, 36). U04S and U08S have proven to be robust tools for a large set of chelating molecules whenever uranyl speciation remains very complex, although currently they do not make it possible to quantify the amounts of various metal species. It is now crucial to investigate the nature of the interaction with uranyl at the paratope sites of mAbs U04S and U08S to devise further applications. A detailed analysis of the energy landscape of the U04S-uranyl interaction has been carried out by atomic force microscopy (37). Crystals of U04S and U08S Fab fragments have recently been obtained, and crystallographic studies are in progress. Acknowledgment. We gratefully thank Yannick Delcuze for skillful assistance in microscopy, Christian Basset for immunochemistry, and Didier Cavadore (Laboratoire d’Analyses Biologiques et Médicales, CEA Marcoule) for ICP-MS measurements. This work was supported by the CEA Program “Toxicologie Nucleaire Environnementale”.

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TX700215E

High-Affinity Uranyl-Specific Antibodies Suitable for Cellular Imaging

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