Aluminum Oxide Nanoparticles as Carriers and ... - ACS Publications

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Aluminum Oxide Nanoparticles as Carriers and Adjuvants for Eliciting Antibodies from Non-immunogenic Haptens Á ngel Maquieira,* Eva M. Brun, Marta Garcés-García, and Rosa Puchades Centro de Reconocimiento Molecular y Desarrollo Tecnológico, Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain S Supporting Information *

ABSTRACT: Functionalized nanomaterials have applications yet to be discovered, especially in the biological field and, in particular, the in vivo production of antibodies. In this paper, we show that aluminum oxide nanoparticles that are covalently coupled to haptens (small molecular mass compounds) activate the immune system, eliciting antibodies (polyclonal and monoclonal) specific for the herbicide atrazine, the antibiotic sulfasalazine, and the vitamin biotin in mice and rabbits. The particles play the role of carrier and adjuvant, with the immune response being dependent on size and crystallinity. The affinity constants of the antibodies are similar to those reached with traditional immunization strategies based on the use of carrier proteins. This approach has not been previously described, being of scientific and practical interest, as it can lead to immunogens safer than the conventional ones, potentially applicable to human vaccination. In addition, the useful advantages of this technique include the stability of the metallic particles, the synthesis of immunogens in organic media, the versatility of particle derivatization, the ease of purification, the full chemical characterization of immunogens, the lack of a requirement for an adjuvant, the reduction of the cross reactivity, and the low cost of materials. Also, the analytical performances of the immunoassays developed using these antibodies are comparable to those obtained by the standard protocols. Analytical applications of the developed ELISAs were fully demonstrated. Characterization (IgG nature, affinity constants, etc.) of the immunoreagents were also fully assessed.

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The preparation of effective hapten−protein conjugates that produce the desired immune response is not always possible or reproducible, which may lead to the unsuccessful synthesis of a product and inconsistent hapten−protein stoichiometries; these inconsistencies result in large variations in antibody yields and properties.7 Moreover, conjugates of proteins with low solubility (KLH or glutamins) precipitate from aqueous media during preparation, leading to problems in dispensing, antigen characterization, and immunization efficiency. Few studies using nonprotein carriers to raise antibodies against small molecules have been undertaken. Arad-Yellin et al.8 obtained antibodies against sulfur mustard and 6-aminohexyl 4-acetoxymethyl benzamide haptens covalently coupled to a carboxylated polysulfone carrier polymer without the use of traditional adjuvants, suggesting the adjuvant nature of polysulfone. Shiosaka et al.9 raised specific, high-titer antibodies against glutamate by immunizing rabbits with glutamate that was bound to a colloidal gold carrier by ionic interactions and van der Waals forces. Following this methodology, specific antibodies against amino acids,10 biotin,11 lysophosphatidic acid,12 and azobenzene dye13 have been observed. Ishii et al.13 have explained that hapten nanoparticles stimulate antibody

anomaterials with new functionalities are demonstrating great potential in areas such as medicine,1 pharmacokinetics,2 and tissue regeneration and repair.3 The application of nanomaterials to immunology, and the in vivo induction of specific antibodies in particular, requires basic and systematic studies. Most importantly, applying nanomaterials involves identifying the immune system’s response to those used as carriers and adjuvants. Protocols for obtaining antibodies from immunization are well-established, especially for high-molecular-weight substances. However, size is critical for the smallest compounds, because they do not provoke an effective immune response, and coupling small molecules to carrier macromolecules is necessary to enable them to activate both B- and T-cells. Landsteiner4 found that small molecules, called “haptens”, could be covalently attached to a carrier protein to produce three distinct sets of antibodies: hapten-specific, carrier-specific, and hapten−carrier conjugate antibodies. In addition to this pioneering coupling strategy, other covalent methods for synthesizing hapten−carrier conjugates have been established.5 Traditionally, haptens are coupled to natural protein carriers, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or thyroglobulin, but the nature of the protein and the protein−hapten conjugate are critical for generating antibodies and increasing their titers. In fact, a recent study6 demonstrated an immunostimulatory change in certain nonimmunogenic proteins after coupling to haptens. © 2012 American Chemical Society

Received: July 24, 2012 Accepted: September 25, 2012 Published: September 25, 2012 9340

dx.doi.org/10.1021/ac3020998 | Anal. Chem. 2012, 84, 9340−9348

Analytical Chemistry

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immunoreagents with similar analytical performances to those developed using the standard methodology.

production by stimulating B-cells without the need for T-cell involvement, because of the suitable concentration of the small chemical and its appropriate size. On the other hand, Dykman and co-workers14 have recently proposed that gold nanoparticles influence the activation of T-cells when used as an antigen carrier. A unique way to elicit an immune response is the production of betatypic anti-idiotypic antibodies, which have the capacity to mimic the original antigen used to generate the primary antibody. He et al.15 have reviewed the application of these antibodies in several immunoassay systems for small molecules. However, the generation of this type of antibody requires the previous generation of primary antibodies from haptens, carriers, and adjuvants, using the traditional immunization method. Other authors have studied the production of antibodies directly from crystalline and amorphous materials. Kam et al.16 established that monosodium ureate monohydrate elicits antibody production in humans and rabbits. Perl-Treves et al.17 also demonstrated that monoclonal antibodies against monohydrate cholesterol crystals specifically recognize those crystals and can selectively distinguish between crystal faces. In addition, Geva et al.18 have shown that monoclonal antibodies raised by immunization with crystals of the tripeptide L-Leu-LLeu-L-Tyr have stereoselective and enantioselective properties that vary depending on the antibody. In brief, these data reveal that nanosize crystalline macromolecular substances trigger a specific immune response. Natural self (alloantigens, i.e., ureate) or foreign substances activate the immune system, showing a response similar to that elicited by traditional immunization conjugates such as proteins, viruses, or microorganisms. The primary difference is that the first group consists of small molecules presented as nanosolids. Consequently, low-molecular-mass compounds anchored on solid carriers should act as effective antigens, generating a specific immune response. Adjuvants, by contrast, are compounds that enhance either the humoral or cytotoxic immune response and are inoculated as a mixture with the hapten conjugates. Traditional adjuvants contain live, attenuated pathogens and inactivated organisms or bacterial toxins in mineral oils.19 The efficacy and safety of different adjuvant formulations were studied,20 and nonbiodegradable nanoparticles such as latex, polystyrene, and gold have been examined as alternative adjuvants.21 These particles remain at the site of injection for extended periods of time, leading to long-lasting presentation and an enhanced immunogenicity of the codelivered antigen.22 Inorganic compounds such as silica and zeolites have shown possible adjuvant activity.23,24 Consequently, a challenging point is the search for better carriers with adjuvant properties. In addition, aluminum hydroxide and aluminum oxide are well-known materials that are used as safe adjuvants in human vaccines. Peptomers covalently linked to aluminum oxide nanoparticles have shown interesting vaccination results against viral pathogens without the use of additional adjuvants.25 Knowing the previous performance of aluminum oxide nanoparticles and that crystals elicit an immune response,16−18 this work hypothesizes that these nanoparticles could be an appropriate carrier and adjuvant for raising antibodies against lowmolecular-mass compounds. Thus, aluminum oxide was chemically derivatized to be covalently conjugated to different hapten targets (the vitamin biotin, the herbicide atrazine and the antibiotic sulfasalazine) and was selected to fully raise

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METHODS The principles of Good Animal Experimentation Practice, as laid down in European Union (EU) legislation, Council Directive 86/609/EEC, were followed in all aspects of the immunization process. Synthesis and Derivatization of Metallic Oxides. The aluminum oxide particles were activated and derivatized with (3-aminopropyl)triethoxysilane, using the method of Frey et al.26 The presence of free amine groups was confirmed with the ninhydrin method.27 Care was taken to avoid contamination with any protein during the derivatization of aluminum oxide. See the Supporting Information and supplementary notes for further details regarding the methods: reagents, SEM and ESIMS measurements, hapten and target data, and the details of the immunization protocols. Immunogen Preparation. Aminated Al2O3 was conjugated to haptens as follows: a solution of dicyclohexylcarbodiimide (DCC) (75 μmol) and N-hydroxysuccinimide ester (NHS) (75 μmol, 8.6 mg), both in anhydrous N,N′dimethylformamide (DMF) (50 μL), was added to the hapten (71.4 μmol dissolved in 70 μL of anhydrous DMF) and stirred for 5 h at room temperature in darkness. After centrifugation, the supernatant was added to a suspension of silanized Al2O3 particles (50 mg) in carbonate buffer, pH 9.6 (1 mL). The reaction mixture was vortexed at room temperature for 2.5 h in darkness and was then stirred overnight at 4 °C. The reaction then was centrifuged, and the supernatant was removed. The particles were washed six times by suspending the pellets in deionized water (1 mL), stirring using a vortex, and centrifuging. The conjugate was dried under a vacuum at room temperature. The quantity of hapten covalently coupled to the particles was determined by elemental analysis. Additional details are described in a patent reference.28 Haptens were covalently conjugated to KLH by activation with DCC.29 The protein concentration in the work steps was determined by the Bradford method. The molar density of the hapten/KLH was difficult to determine by MALDI-TOF-MS, because of the high molecular weight of this protein. It was also not possible to determine this ratio spectrophotometrically, because of the overlap between the protein absorption band (280 nm) and the maximum absorption band of the haptens used. The covalent coupling of haptens to aprotinin was performed by following the procedure described previously.29 The conjugates were purified by dialysis against PBS (10 mM phosphate buffer, 137 mM NaCl, and 2.7 mM KCl, pH 7.4). The aprotinin−hapten conjugates were covalently coupled to aluminum nanoparticles using the following procedure. A solution of the aprotinin−hapten conjugate (1 mL) containing 2.5 mg of this protein was mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (5 mg). After homogenization, aminated Al2O3 particles (50 mg) were added, and the mixture was vortexed for 3 h. The solution was then centrifuged (7500 g) to remove the supernatant. The solid was washed six times by resuspending the pellets in deionized water (1 mL) and aspirating the supernatant. Finally, the particles were suspended in 1 mL of PBS and stored at −20 °C. ELISA Procedure. First, the avidity of sera against the coating conjugates (OVA linked to the same hapten used for immunization) was determined on a noncompetitive indirect 9341



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ELISA format. Indirect competitive ELISA assays were carried out with those sera showing specific recognition. In brief, flat-bottomed polystyrene ELISA plates were coated with 100 μL/well of the appropriate concentration of the OVA−hapten conjugate solution in carbonate buffer (50 mM carbonate−bicarbonate buffer, pH 9.6). The plates were then incubated overnight at 4 °C. The following day, plates were washed six times with PBS-T (PBS containing 0.05% Tween 20). For competitive assays, a volume of 50 μL of the appropriate sera dilution (in 2-fold concentrated PBS-T) and 50 μL of standards in deionized water were added to the coated plates and incubated for 1 h at room temperature. After being washed as earlier, plates were incubated for 1 h with peroxidaselabeled goat antirabbit immunoglobulins (GAR-HRP) or peroxidase-labeled goat antimouse diluted 1:4000 and 1:2000, respectively, in PBS-T (100 μL/well). Peroxidase activity was determined by adding 100 μL/well of substrate solution (2 mg/ mL OPD and 0.012% H2O2 in 25 mM sodium citrate and 62 mM sodium phosphate, pH 5.5). After 10 min, the enzymatic reaction was stopped by adding 2.5 M H2SO4 (50 μL/well), and the absorbance was read in dual-wavelength mode (490 nm as the test wavelength and 650 nm as the reference wavelength).

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RESULTS AND DISCUSSION Aluminum Oxide Nanoparticles as Functional Immunization and Adjuvant Materials for Small Molecules. To evaluate the potential of aluminum oxide nanoparticles as a carrier for immunization, we first derivatized Al2O3 by silanization to provide them with a terminal amine group. The amination percentage was 2%, which is similar to the theoretical proportion of primary amines for BSA (1.2%)30 and higher than the theoretical proportion for ovalbumin (OVA) (2). In addition, the immunization kinetics in mice are more regular than those in rabbits. Most importantly, the presence of protein or adjuvant does not offer advantages with respect to immunization with Al2O3 nanoparticles alone. ELISA Characteristics of the Obtained Immunoreagents. Indirect competitive protein−hapten coated ELISA plate assays were carried out with those sera showing specific recognition (Figure 5). The calibration curves in rabbits showed that a high sensitivity was obtained when a protein was present in the immunogen. This result could be due to the coating conjugates used in the ELISA, which are optimized for sera obtained from protein immunogens and not aluminum. On the other hand, the adjuvant seems to play a determining role in the sensitivity. Immunization with the nanoparticle−hapten immunogen provided a lower sensitivity than that exhibited from the traditional protocol (see Figure 5d). Nevertheless, this parameter is acceptable and could be improved by optimizing the assay. In mice, the sensitivity to the Al2O3−hapten improved notably when FA was used (see Figures 5a and 5c). However, the sera from the Al2O3−aprotinin−hapten without FA animals displayed good sensitivity and IC50s for atrazine and sulfasalazine that were comparable to those obtained by the traditional immunization procedure (for example, KLH− hapten and FA). The opposite trend was observed for biotin. Moreover, no competition was achieved with the sera from the Al2O3−biotin hapten animals, with or without FA. This result suggests that the antibodies obtained from these immunogens had a much higher affinity for the hapten than the analyte, because biotin was not able to displace the antibodies from the coating conjugate−antibody immunocomplex. Comparing the results between species revealed that the competitive assays obtained from rabbits were more sensitive than those obtained from mice and especially with protein immunogens (see Table 1). The behaviors of each type of immunogen are different, depending on the target, indicating that the chosen hapten modulates the response and plays an important role in the activation of the immune system. Nevertheless, it must be noted that these are preliminary results and that sensitivity could be improved by optimizing the ELISA conditions. The effect of size and crystallinity was shown in the competitive curves, corresponding to the sera from the 40-nm Al2O3−2d conjugates, which had a lower, but similar, sensitivity as the sera from 300-nm particles (see Figure S6 in the Supporting Information). The interaction kinetics between the HRP−2d conjugate and the rabbit antibodies obtained (with adjuvant) from several immunogens (KLH−2d, Al2O3−2d, and Al2O3−aprotinin−2d) were studied by dual-polarization interferometry (DPI) after being immobilized on a silicon oxynitride chip surface. This immobilization allowed the dissociation constant to be evaluated based on measurements of the phase changes of the antibody−antigen complexes in the transverse magnetic and transverse electric polarization modes.32 The KD values obtained were 3.2 × 10−6 M, 3.0 × 10−5 M, and 1.1 × 10−5 M for antibodies specific for KLH−2d, Al2O3−2d, and Al2O3− aprotinin−2d, respectively. These values indicate that the three

Table 1. Sensitivity of the Antibodies Derived from Different Immunogens in Mice and Rabbits IC50 (ng/mL)

a

immunogen

mice

KLH−2d* Al2O3−2d* Al2O3−2d Al2O3−aprot−2d* Al2O3−aprot−2d Aprot−2d* KLH−B* Al2O3−B* Al2O3−B Al2O3−aprot−B* Al2O3−aprot−B Aprot−B* KLH−S* Al2O3−S* Al2O3−S Al2O3−aprot−S* Al2O3−aprot−S Aprot−S*

270 ± 18 609 ± 32 1182 ± 156 2255 ± 287 689 ± 45 1564 ± 164 175 ± 15 n.c.a n.c.a 162 ± 13 2586 ± 252 742 ± 51 1.5 ± 0.1 130 ± 10 1592 ± 126 2215 ± 203 6.1 ± 0.8 43 ± 5

rabbits 1.6 274 260 5.7 32 1.1

± ± ± ± ± ±

0.2 14 11 0.6 4 0.1

n.c. = no competition.

antibodies bound the tracer HRP−2d with similar affinity. Although the antibodies from the KLH−2d immunogen showed a binding constant 10 times greater than those of the other sera, this value matches the sensitivity (IC50) obtained from the indirect competitive ELISA assays (Table 1). Serum Selectivity. The selectivity of the rabbit sera for atrazine was studied using indirect competitive assays with different s-triazines. Three sera were compared: one obtained from the traditional immunization protocol (KLH−2d plus adjuvant), and two using Al2O3−2d as the immunogen, with or without Freund’s adjuvant. The only compound that showed high cross-reactivity in any of the sera was propazine (see Table S2 in the Supporting Information ). However, the serum from the Al2O3−2d with adjuvant sample showed a higher selectivity against propazine, compared to the sera obtained from immunization with KLH−2d. High cross-reactivity with propazine is a common behavior of the classical polyclonal antiatrazine antibodies obtained from haptens such as 2d.33 Thus, this result is notable and promising for obtaining sera with different properties. On the other hand, this serum had a lower cross-reactivity with simazine and a higher cross-reactivity with prometryn than the other sera, which all showed similar results. This result suggests that the KLH−2d immunogen might be presented differently to T-cells, thus producing antibodies with another recognition capacity. The In Vivo Adsorption of Host Protein by Al2O3. The rabbit antibodies obtained by immunization with Al2O3−2d without a protein intermediate were mainly of the IgG isotype, indicating a T-cell-dependent immune response. This type of immune response is directly related to protein antigens. Because we have used nonprotein antigens, this result could be explained if the aluminum oxide particles are able to covalently bind34−36 or adsorb to host proteins to reach the Tcells. To investigate this hypothesis, we carried out several in vitro experiments investigating the protein adsorption capacity of aluminum particles. For these experiments, the original oxide, the activated form, the silanized form, and the oxide form 9345



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and especially proteins is, in practice, reduced to the free amine group of lysines and a few other groups.7 In addition, hapten−oxide conjugation can be carried out in organic solvents, which is interesting because haptens are frequently apolar compounds. High salt concentrations and broad pH ranges used in biomolecule management can be assayed in aqueous media, thus allowing the use of a large collection of well-established organic synthesis methods, sharply improving the coupling yield. Nevertheless, in this work, haptens have been coupled to a metallic oxide using the standard protein−hapten coupling method (dicyclohexylcarbodiimide in dimethylformamide/carbonate buffer) to compare our results with the established linking protocols. The easy purification and high stability of this method, compared to protein methods, must also be noted. We have developed a systematic antigen preparation method to obtain well-characterized, reproducible immunogens for haptens using an inexpensive, solid nanosupport prepared in a simple and reproducible way (see Table S3 in the Supporting Information). The approach could be broadly applied to immunization purposes, especially when hapten−protein coupling is difficult. Moreover, we cannot control the immunogen preparation steps, such as the hapten:protein ratio, the mode of presentation, the immunogen quantity or doses with the traditional methodology. In addition, the chemical characterization of the conjugates is difficult, because techniques such as MALDI-TOF-MS are unavailable and the hapten:protein ratio is often inaccurate, especially when the protein carrier has a high molecular weight.

coupled to a hapten were stirred in a BSA solution. The different samples were washed and successively centrifuged to remove protein residues until no protein was detected in the supernatant. The oxides then were subjected to denaturing electrophoresis (Figure 6).

Figure 6. Protein adsorption capacity of the aluminum particles. Gel electrophoresis of the supernatants: lane 1, Al2O3; lane 2, activated Al2O3; lane 3, silanized Al2O3; lane 4, Al2O3−2d; and lane 5, BSA.

All of the samples showed the presence of BSA, which could explain the robust immune response obtained when Al2O3 particles were used as a carrier. It is notable that the Al2O3−2d conjugate (run 4 in Figure 6) shows more protein adsorption than the other samples, although we have not found a logical explanation for this finding. Thus, in vitro gel electrophoresis demonstrated that the aluminum oxide particles should be able to adsorb to host proteins in vivo. The adsorption of proteins onto aluminum salts has been widely studied,37 suggesting that the destabilization of the adsorbed proteins is the mechanism of action for this adjuvant. New experimental work could be developed to shed more light on and support our in vivo results. These results open a broad line of research into the use of aluminum oxide nanoparticles as secure, inexpensive, and potent carriers for haptens and for the development of the safest immunogens for low-molecular-weight compounds. For instance, a cocaine vaccination study38 used a protein derived from the cholera B toxin as a carrier and administered the vaccine with an aluminum adjuvant. The study of nanoparticulated nonprotein carriers for this particular vaccination strategy should be of interest. Thus, our approach would be key for developing skin immunization treatments for small molecules, as has been demonstrated for proteins such as hepatitis B surface antigen absorbed in aluminum oxide.39 Nanoparticulated aluminum oxide presents many advantages as a carrier. First, it is nontoxic, and it is one of (along with monophosphoryl lipid A) only two adjuvants currently licensed for addition to human vaccines in the United States.40 The chemical stability and low price of nanoparticulated aluminum oxide and monophosphoryl lipid A, in comparison to that of proteins, must also be highlighted. Furthermore, aluminum oxide can be functionalized by different chemistries with variable spacer arm length. These attributes are important because the variety of functional groups allows conjugation to haptens with very different binding moieties. In contrast, the potential theoretical binding group for biomolecular carriers

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CONCLUSION In this work, it is demonstrated that low-molecular-weight compounds that are directly attached to aluminum oxide particles are potent immunogens that induce humoral responses. Nanoparticles, especially 300-nm particles, are more favorable for immunizing mice than microparticles. This size is believed to be optimal for mucosal immunization purposes,26 because it is within the range described in the literature for vaccines.41 The results show that covalent linkage is essential for the efficient induction of immune responses, as inoculation with free hapten or a mixture of hapten and particles induced no response. To our knowledge, this study is the first attempt to use Al2O3 as a direct carrier for small hapten immunization. The evidence supporting the activation of the immune system by presenting the hapten after adsorbing its own proteins consists of very interesting scientific and practical results. The hypothesized explanation given in this paper is in accordance with the old “dogma” regarding the activation and mechanisms for eliciting hapten-specific antibodies at the cellular level. However, recent studies have reported that the complement system is activated by nanoparticles containing hydroxyl, amine, or thiol on their surface, yielding C3 nanoparticle covalent bonds that finally trigger the immune system.40 Another hypothesis is the activation of macrophages and lymphocytes when nanoparticles are used as antigen carriers.8 Recent research has noted that the DNA that is released from dying host cells mediates aluminum adjuvant activity.42 These alternative explanations also support our experimental results. From an analytical view, the newly developed reagents showed affinity constants, selectivity, and sensitivity comparable with that obtained by the standard immunization and ELISA 9346



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(10) Shiosaka, S.; Kono, J.; Kiyama, H.; Tohyama, M.; Shiotani, Y. Antibodies specific to low molecular-weight substances attached to metal colloidal particles. Jpn. Patent JP 62162963 A 19870718, 1987. (11) Dykman, L. A.; Matora, L. Y.; Bogatyrev, V. A. J. Microbial. Meth. 1986, 24, 247−248. (12) Chen, J.; Zou, F.; Wang, N.; Xie, S.; Zhang, X. Bioorg. Med. Chem. Lett. 2000, 10, 1691−1693. (13) Ishii, N.; Fitrilawati, F.; Manna, A.; Akiyama, H.; Tamada, Y.; Tamada, K. Biosci. Biotechnol. Biochem. 2008, 72, 124−131. (14) Dykman, L. A.; Staroverov, S. A.; Bogatyrev, V. A.; Shchyogolev, S. Y. Gold Nanoparticles: Properties, Characterization and Fabrication; Chow, P. E., Ed.; Nova Science Publishers: Hauppauge, NY, 2010; pp 59−88. (15) He, J.; Fan, M.-T.; Liang, Y.; Liu, X.-J. Chin. J. Anal. Chem. 2010, 38, 1366−1370. (16) Kam, M.; Perl-Treves, D.; Caspi, D; Addadi, L. FASEB 1992, 6, 2608−2613. (17) Perl-Treves, D.; Kessler, N.; Izhaky, D.; Addadi, L. Chem. Biol. 1996, 3, 567−577. (18) Geva, M.; Frolow, F.; Eisenstein, M.; Addadi, L. J. Am. Chem. Soc. 2003, 125, 696−704. (19) Salvador, A.; Igartua, M.; Hernández, R. M.; Pedraz, J. L. J. Drug Delivery 2011, DOI: 10.1155/2011/181646. (20) Morefield, G. L. AAPS J. 2011, 13, 191−200. (21) Kalkanidis, M.; Pietersz, G. A.; Xiang, S. D.; Mottram, P. L.; Crimeen-Irwin, B.; Ardipradja, K.; Plebanski, M. Methods 2006, 40, 20−29. (22) Brunner, R.; Jensen-Jarolim, E.; Pali-Schöll, I. Immunol. Lett. 2010, 128, 29−35. (23) Harkema, J. R.; Rowley, N.; Bramble, L.; Zhang, Q.; Baker, G.; Jackson-Humbles, D.; Wagner, J.; Worden, R. M. Am. J. Respir. Crit. Care Med. 2011, 183, A2282. (24) Danilczuk, M.; Dlugopolska, K.; Ruman, T.; Pogocki, D. Mini Rev. Med. Chem. 2008, 8, 1407−1417. (25) Frey, A.; Mantis, N.; Kozlowski, P. A.; Quayle, A. J.; Bajardi, A.; Perdomo, J. J.; Robey, F. A.; Neutra, M. R. Vaccine 1999, 17, 3007− 3019. (26) Frey, A.; Neutra, M. R.; Robey, F. A. Bioconjugate Chem. 1997, 8, 424−433. (27) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 117, 147−157. (28) Brun, E. M.; Garcés-García, M.; Maquieira, A.; Puchades, R. Method for producing antibodies by immunization with haptens bound to metal oxide particles. World Patent WO2008068359, 2008. (29) Brun, E. M.; Garcés-García, M.; Puchades, R.; Maquieira, A. J. Immunol. Meth. 2004, 295, 21−35. (30) Kublashivili, R. I.; Ugrekhelidze, D. S. Chem. Nat. Compd. 2005, 41, 340−343. (31) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Harlow, E. , Lane, D., Eds.; Cold Spring Harbor Laboratory Press: New York, 1988; pp 92−114. (32) Lin, S.; Lee, C.-K.; Lin, Y.-H.; Lee, S.-Y.; Sheu, B.-C.; Tsai, J.-C.; Hsu, S.-M. Biosens. Bioelectron. 2006, 22, 715−721. (33) González-Martínez, M. A.; Puchades, R.; Maquieira, A.; Ferrer, I.; Marco, M. P.; Barceló, D. Anal. Chim. Acta 1999, 386, 201−210. (34) Sim, R. B.; Wallis, R. Nat. Nanotechnol. 2011, 6, 80−81. (35) Hamad, I.; Al-Hanbali, O.; Hunter, A. C.; Rutt, K. J.; Andresen, T. L.; Moghimi, S. M. ACS Nano 2010, 4, 6629−6638. (36) Salvador-Morales, C.; Zhang, L.; Langer, R.; Farokhzad, O. C. Biomaterials 2009, 30, 2231−2240. (37) Jones, L. S.; Peek, L. J.; Power, J.; Markham, A.; Yaziie, B.; Middaugh, C. R. J. Biol. Chem. 2005, 280, 13406−13414. (38) Martell, B. A.; Orson, F. M.; Poling, J.; Mitchell, E.; Rossen, R. D.; Gardner, T.; Kosten, T. R. Arch. Gen. Psychiatry 2009, 66, 1116− 1123. (39) Maa, Y. F.; Ameri, M.; Rigney, R.; Payne, L. G.; Chen, D. Pharm. Res. 2004, 21, 515−523. (40) Schijns, V. E. J. C.; Lavelle, E. C. Expert Rev. Vaccines 2011, 10, 539−550.

development reference, with the important advantages of no cross-reaction with the carrier protein, ability to work in organic media, and the use of organic synthesis approaches that cannot be considered with proteins.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information: X-ray diffraction of the Al2O3 particles used; ESI-MS for the aprotinin−2d and aprotinin−biotin conjugates; scheme for the synthesis, purification, and characterization of the immunogens studied; comparative antibody titers obtained after the immunizations of rabbits and mice with different antigens; the competitive assay curves obtained using antibodies generated in response to different size Al2O3 nanoparticles coupled to 2d hapten with or without Freund’s adjuvant in mice; final mouse serum mean absorbance values (λ = 490 nm); cross-reactivity of the rabbit sera with atrazine-related compounds; characterization techniques for the chemical species and conjugates; reagent, hapten and target data, SEM, ESI-MS, and X-ray measurements, immunization protocols, morphological changes in mice, determination of affinity constants by dual-polarization interferometry and electrophoretic adsorption assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +34 96 3877342. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful to S. Gee (Entomology Department, UC Davis, CA, USA) for aiding previous exploratory work, to T. Gorris and M. Cambra (IVIA, Valencia, Spain) for managing the animal experiments and obtaining the monoclonal antibodies, to M. Plebanski (Department of Immunology, Monash University, Australia) for his valuable aid in planning the research, and to G. González-Sapienza (Department of Immunology, Universidad de la República, Montevideo, Uruguay) for the sharp recommendations and critical evaluation of the results. This research was partially supported by Prometeo 2010/008 (Generalitat Valenciana) and FEDER MICINN CTQ2010-15943 project.

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