Development of an Enzyme-Linked Immunosorbent Assay for the

Anal. Chem. 2006, 78, 71-81

Development of an Enzyme-Linked Immunosorbent Assay for the Determination of the Linear Alkylbenzene Sulfonates and Long-Chain Sulfophenyl Carboxylates Using Antibodies Generated by Pseudoheterologous Immunization Javier Ramo´n-Azco´n, Roger Galve, Francisco Sa´nchez-Baeza, and M.-Pilar Marco*

Applied Molecular Receptors Group (AMRg). Department of Biological Organic Chemistry. IIQAB-CSIC. Jorge Girona, 18-26, 08034-Barcelona, Spain

ELISA methods have been developed for screening contamination of water resources by linear alkyl benzene sulfonates (LAS) or the most immediate degradation products, the long chain sulfophenyl carboxylates, SPCs. The assay uses antibodies raised through pseudoheterologous immunization strategies using an equimolar mixture of two immunogens (SFA-KLH and 13C13-SPCKLH) prepared by coupling N-(4-alkylphenyl)sulfonyl-3aminopropanoic acid (SFA) and p-(1-carboxy-13-tridecyl)phenylsulfonic acid (13C13-SPC) to keyhole limpet hemocyanin (KLH). The immunizing haptens have been designed to address recognition versus two different epitopes of the molecule. The SFA hapten maximizes recognition of the alkyl moiety while preserving the complexity of the different alkyl chains present in the LAS technical mixture. The 13C13-SPC hapten addresses recognition of the common and highly antigenic phenylsulfonic group. The antisera raised using this strategy have been shown to be superior to those obtained through homologous immunization procedures using a single substance. By using an indirect ELISA format, LAS and long-chain SPCs can be detected down to 1.8 and 0.2 µg L-1, respectively. Coefficients of variation of 6 and 12% within and between assays, respectively, demonstrate immunoassay reproducibility. The assay can be used in media with a wide range of pH and ionic strength values. Preliminary experiments performed to assess matrix effects have demonstrated the potential applicability of the method as a screening tool to assess contamination by these types of surfactants in natural water samples.

INTRODUCTION Linear alkylbenzene sulfonates (LAS) are a mixture of closely related isomers and homologues, each containing an aromatic ring sulfonated at the para position and attached to a linear alkyl chain. LAS are one of the major anionic surfactants used on the market * To whom correspondence should be sent. Phone: 93 4006171. Fax: 93 2045904. E-mail: [email protected]. 10.1021/ac051141s CCC: $33.50 Published on Web 11/25/2005

© 2006 American Chemical Society

today in cleaning products for home, institutional, and industrial use, for example, car wash liquids, laundry detergents, liquid dish detergents, hard surface cleaners, dry cleaning products, and waterless hand and industrial cleaners. The European consumption of LAS was estimated to be ∼400 Kton in year 2000, and the global consumption is around 1.6 million tons/year.1 As a consequence of this extensive use, residues of LAS and their degradation products are found in almost all types of environmental water and soil samples near urban and industrial areas at significant concentration levels.2 As an example, the daily mass input of LAS and their biodegradation intermediates from the Sancti Petri Channel to Cadiz Bay was 44.6 kg.3 In a study of the year 20024 in Rio Macacu (Brazil), LAS concentrations ranged between 14 and 155 µg L-1, and for sulfophenyl carboxylic (SPCs) degradation products, from 1.2 to 14 µg L-1. Similarly, in Europe, the levels of SPCs in the Llobregat (Spain) and Rhine (Germany) Rivers amounted to 5 and 1.8 µg L-1, respectively. Treatments in the corresponding waterworks produced drinking water with SPC levels around 2 µg L-1 (Spain) and 0.05 µg L-1 (Germany).5 LAS are not especially toxic by themselves, but because of their amphoteric structure, they can contribute to the permeation of other pollutants (i.e., heavy metals and pesticides) through biological membranes into aquatic organisms.6,7 Moreover, these chemicals degrade rapidly aerobically, whereas they do not degrade under anaerobic conditions. Therefore, there is a risk of bioaccumulation in aquatic plants and organisms. Their total biodegradation still requires 5-10 days under normal conditions.1 The first degradation products are long-chain sulfophenyl carbox(1) Perales, J. A.; Manzano, M. A.; Sales, D.; Quiroga, J. M. Bull. Environ. Contam. Toxicol. 1999, 63, 94-100. (2) Petrovic, M.; Barcelo, D. In Analysis and Fate of Surfactants in the Aquatic Environment; Knepper, T. P., Barcelo, D., de Voogt, P., Eds.; Elsevier: Amsterdam, 2003; Vol. XL, pp 655-674. (3) Leon, V. M.; Saez, M.; Gonzalez-Mazo, E.; Gomez-Parra, A. Sci. Total Environ. 2002, 288, 215-226. (4) Eichhorn, P.; Rodrigues, S. V.; Baumann, W.; Knepper, T. P. Sci. Total Environ. 2002, 284, 123-134. (5) Eichhorn, P.; Knepper, T. P.; Ventura, F.; Diaz, A. Water Res. 2002, 36, 2179-2186. (6) Blasco, J.; Gonza´lez-Mazo, E.; Sasquete, C. Toxicol. Environ. Chem. 1999, 71, 447-456. (7) Lewis, M. A. Water Res. 1992, 26, 1013-1023.

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ilic acids,8 formed via ω-oxidation of the alkyl chains. Subsequent β-oxidation steps result in congeners with shorter alkyl chains, some of them extremely polar, that may remain in the environment, since further degradation of the aromatic ring occurs slowly. The analytical determination of LAS in environmental samples has relied on the use of chromatographic and spectrometric techniques, such as liquid chromatography/mass spectrometry (LC/MS).9-11 The use of gas chromatography/mass spectrometry (GC/MS) has also been reported,12 but it involves a derivatization step. Cleanup/preconcentration steps consisting of liquid-liquid extraction with organic solvents followed by solid-phase extraction (SPE) procedures are always required. Their ability to adsorb to the solid surfaces, through their hydrophobic side, prevents representative results of real environmental concentrations.13 Moreover, the complexity of the chromatograms obtained may also make routine screening and multiple analyses difficult. As an effective alternative, immunochemical techniques could not only afford the necessary detectability and specificity for the target analyte but also offer other advantages, such as reliability, simplicity, low cost, and high sample throughput capabilities.14-19 The preparation of optimum haptens as immunogens and competitors has been regarded as the most crucial step in the development of an immunochemical technique for small molecules. Many literature examples prove that an appropriate hapten design determines the features of the resulting antibodies, which mainly govern the specificity and the selectivity of an immunochemical technique.14,20-23 Theoretical molecular models and calculations can be useful tools to assist prediction of which hapten will be the most appropriate to raise antibodies;24-26 however, LAS exhibit the particularity of being a complex mixture of substances (8) Scho ¨berl, P. Tenside, Surfactants, Deterg. 1989, 26, 2. (9) Hites, R. A. Biotherapy (Dordrecht, Neth.) 1998, 11, 77-96. (10) Scullion, S. D.; Clench, M. R.; Cooke, M.; Ashcroft, A. E. J. Chomatogr. 1996, 733, 207-216. (11) Petrovic, M.; Barcelo´, D. Anal. Chem. 2000, 72, 4560-4567. (12) Trehy, M. L. Anal. Chem. 1990, 62, 2581-2586. (13) Marcomini, A.; Di Corcia, A.; Samperi, R. Environ. Sci. Technol. 1994, 28, 850-858. (14) Gabaldon, J. A.; Maquieira, A.; Puchades, R. Crit. Rev. Food Sci. Nutr. 1999, 39, 519-538. (15) Fitzpatrick, J.; Fanning, L.; Hearty, S.; Leonard, P.; Manning, B. M.; Quinn, J. G.; O’Kennedy, R. Anal. Lett. 2000, 33, 2563-2609. (16) Harris, A. S.; Wengatz, I.; Wortberg, M.; Kreissig, S. B.; Gee, S. J.; Hammock, B. D. Mult. Stresses Ecosyst. 1998, 135-153. (17) Tang, Z.; Karnes, H. T. Biomed. Chromatogr. 2000, 14, 442-449. (18) Sherry, J. Chemosphere 1997, 34, 1011-1025. (19) Oubin ˜a, A.; Ballesteros, B.; Bou-Carrasco, P.; Galve, R. G., J.; Iglesias, F.; Sanvicens, N.; Marco, M. P. In Sample Handling and Trace Analysis of Pollutants.; Barcelo´, D., Ed.; Elsevier: Amsterdam, 2000; pp 1075-1101. (20) Skerritt, J. H.; Lee, N. ACS Symp. Ser. 1996, 124-149. (21) Lawruk, T. S.; Hottenstein, C. S.; Fleeker, J. R.; Rubio, F. M.; Herzog, D. P. In Herbicide Metabolites in Surface Water and Groundwater; Meyer, M. T., Thurman, E. M., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996; Vol 630. (22) Goodrow, M. H.; Sanborn, J. R.; Stoutamire, D. W.; Gee, S. J.; Hammock, B. D. In Immunoanalysis of Agrochemicals. Emerging Technologies; Nelson, J. O., Karu, A. E., Wong, R. B., Eds.; American Chemical Society: Washington, DC, 1995. (23) Carlson, R. E. In Immunoanalysis for Agrochemicals; Nelson, J. O., Karu, A. E., Wong, R. B., Eds.; American Chemical Society: Washington, DC, 1995; pp 141-152. (24) Ballesteros, B.; Barcelo´, D.; Sanchez-Baeza, F.; Camps, F.; Marco, M. P. Anal. Chem. 1998, 70, 4004-4014. (25) Lee, N. J.; Skerritt, J. H.; McAdam, D. P. J. Agric. Food Chem. 1998, 46, 1730-1739. (26) Galve, R.; Camps, F.; Sanchez-Baeza, F.; Marco, M.-P. Anal. Chem. 2000, 72, 2237-2246.

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(different alkyl chain lengths and positional isomers), which makes antibody production even more challenging. Fujita et al. have reported the preparation of antibodies using 5-(4-sulfophenyl)pentanoic acid, a single substance with a short alkyl chain, as an immunizing hapten.27 This chemical structure addressed antibody recognition versus the sulfonic group, which is one of the most antigenic determinants in this molecule; however, studies performed in our group indicate that the contribution of the alkyl chain in the stabilization of the antibody-analyte should not be underestimated.28 In the field of catalytic antibodies, several research groups have made use of heterologous immunization strategies to elicit antibody immunoresponse against two different epitopes of the molecules, simplifying hapten synthesis.29-31 Thus, although an ideal hapten preserving both groups would have a spacer arm at the ortho or meta positions, the synthetic pathway leading to these types of chemical structures could be troublesome. The heterologous immunization strategy devised by the group of Prof. Masamune31-33 consists of successfully immunizing the animal with two different but structurally related haptens. It has mainly been used for zwirterionic transition-state analogues with positive and negative charges, for which chemical preparation of the corresponding hapten could be problematic. Instead, the heterologous immunization using two individual haptens containing a different charge provided an opportunity to simultaneously generate an acidic and a basic catalytic residue in the antibody combining site. Thus, Ersoy et al.,29,34 designed three haptens to produce, by heterelogous immunization, nucleophile-mediated (phenol) amide bond cleaving catalytic antibodies having a binding pocket with (i) a hydrophobic area, (ii) an acidic residue complementary to the oxyanionic transition state, and (iii) a basic residue to aid deprotonation of a phenol nucleophile and protonation of the departing amine. In all those examples, the catalytic activity of the antibodies generated through heterologous strategies is higher than those obtained by homologous immunization. Attending to these precedents, the present paper describes for the first time the use of this immunization approach to obtain antibodies against an amphoteric molecule such as LAS with two well differentiated epitopes in its chemical structure. The results show that the antibodies obtained by applying this strategy provide better immunoassays than those obtained through homologous immunization strategies. EXPERIMENTAL SECTION Chemistry. General Methods and Instruments. Thin-layer chromatography (TLC) was performed on 0.25-mm, precoated, silica gel 60 F254 aluminum sheets (Merck, Darmstadt, Germany). (27) Fujita, M.; Ike, M.; Goda, Y.; Fujimoto, S.; Toyoda, Y.; Miyagawa, K.-I. Environ. Sci. Technol. 1998, 32, 1143-1146. (28) Estevez, M.-C.; Kreuzer, M.; Sanchez-Baeza, F.; Marco, M.-P. Environ. Sci. Technol., in press. (29) Ersoy, O.; Fleck, R.; Blanco, M. J.; Masamune, S. Bioorg. Med. Chem. 1999, 7, 279-286. (30) Reymond, J. L. In Biocatalysis - from Discovery to Application; SpringerVerlag Berlin: Berlin 33, 1999; Vol. 200, pp 59-93. (31) Tsumuraya, T.; Suga, H.; Meguro, S.; Tsunakawa, A.; Masamune, S. J. Am. Chem. Soc. 1995, 117, 11390-11396. (32) Suga, H.; Ersoy, O.; Williams, S. F.; Tsumuraya, T.; Margolies, M. N.; Sinskey, A. J.; Masamune, S. J. Am. Chem. Soc. 1994, 116, 6025-6026. (33) Tsumuraya, T.; Takazawa, N.; Tsunakawa, A.; Fleck, R.; Masamune, S. Chem.sEur. J. 2001, 7, 3748-3755. (34) Ersoy, O.; Fleck, R.; Sinskey, A.; Masamune, S. J. Am. Chem. Soc. 1998, 120, 817-818.

Figure 1. Schemes showing the synthetic pathways used to prepare immunizing (SFA and 13C13-SPC) and competitor haptens.

Unless otherwise indicated, purification of the reaction mixtures was accomplished by “flash” chromatography using silica gel as the stationary phase. 1H and 13C NMR spectra were obtained with a Varian Unity-300 (Varian Inc., Palo Alto, CA) spectrometer (300 MHz for 1H and 75 MHz for 13C). The chemical reagents used in this synthesis were obtained from Aldrich Chemical Co. (Milwaukee, WI). The mixture of alkylbenzene synthetic precursors of LAS was kindly provided by PETRESA S.A. (San Roque, Ca´diz, Spain). Synthesis of the SFA (Sulfonamide) Hapten. (See scheme 1 in Figure 1) Spectroscopic and spectrometric data are given as Supporting Information. N-(4-Alkylphenyl)sulfonyl-3-aminopropanoic Acid (SFA). Chlorosulfonic acid (1.7 mL, 25 mmol, 3 equiv) was placed in a two-neck round-bottom flask provided with magnetic stirring and under Ar atmosphere. One of the necks was connected to a trap with 1 M NaOH to neutralize the HCl formed. The other neck was used to add slowly the alkylbenzene technical mixture (1 g, 4.2 mmol) at room temperature. The initial pale yellow color of the reaction mixture became intense red. The reaction was finished after 1:30 h, as observed by TLC (hexane as mobile phase). The crude mixture was poured over a water-ice mixture (100 mL), and the white precipitate formed was extracted with hexane, washed with saturated NaHCO3, dried with anhydrous MgSO4, filtered, and evaporated. The crude residue was then purified by silica gel flash chromatography using hexane as mobile phase to obtain the corresponding mixture of 4-alkylphenylsulfonyl chlorides as a pale yellow oil (400 mg, 30% yield). A solution of the mixture of 4-alkylphenylsulfonyl chlorides (200 mg, 0.6 mmol) in anhydrous CH2Cl2 (2 mL) was slowly added under Ar atmosphere to a solution of triethylamine (190 mg, 1.4 mmol, 2.3 equiv), and the chlorhydrate of methyl 3-aminoproanoate (107 mg, 0.77 mmol, 1.3 equiv) in the same solvent (3 mL) was placed in a threeneck round-bottom flask with magnetic stirring. The mixture was left to react at RT until the starting material disappeared by TLC (hexane/ethyl acetate, 1:1). The solvent was evaporated, suspended in saturated NaHCO3, and extracted with EtAcO (ethyl

acetate). The organic layer was dried with MgSO4, filtered, and evaporated to dryness to obtain a mixture of methyl N-(4alkylphenyl)sulfonyl-3-aminopropanoates (140 mg, 60% yield) as a yellow oil. Finally, the esters were hydrolyzed in MeOH (1.3 mL) with 1 M KOH (1 mL, 1 mmol, 3 equiv) for 2 h. The solvent was then evaporated, and the residue was dissolved with 1 N HCl and extracted with EtAcO to obtain SFA (90 mg, 67% yield) as a yellow oil. Synthesis of SPC Haptens. Different SPCs (5C5, 6C6, 7C7, 9C9, 12C12 and 13C13) were synthesized from the corresponding ω-phenylalkylcarboxylic acids 1b-6b by sulfonation of the aromatic ring. The 5-phenylpentanoic acid 1b and the (4carboxybutyl)triphenylphosphonium bromide used to prepare 2a were obtained from commercial sources. The phenylcarboxylic acids 2b-6b were prepared through a Wittig reaction using benzaldehyde and the respective phosphonium bromide salts 2-6, followed by reduction of the double bond formed with H2 using Pd/C as catalyst (see scheme 2 in Figure 1). A general procedure is described below. Synthetic details and spectroscopic and spectrometric data for each particular compound are provided as Supporting Information. Preparation of (ω-Carboxyalkyl)triphenylphosphonium Bromides 2-6. General Protocol. The corresponding ω-bromoalkanoic acid (9 mmol, 1 equiv) and the triphenylphosphine (9 mmol, 1 equiv) were placed in a round-bottom flask equipped with a cooling jacket and a magnetic stir bar. The mixture was heated to 100 °C under Ar atmosphere. After 4 h, a total conversion of the acid was observed by 1H NMR. The reaction was left overnight under vacuum at 100 °C. The yield was quantitative. Preparation of ω-Phenylalkylcarboxylic Acids 1b-6b. General Protocol. Step 1: Synthesis of the Methyl ω-Phenylalkenoates 1a-6a. Anhydrous DMSO (6.1 mL, 0.1 mol, 3.5 equiv) was added to a suspension of 60% NaH (1.24 g, 0.3 mol, 2.1 equiv; previously washed with anhydrous pentane (3 × 15 mL) and dried), placed in a round-bottom flask equipped with a magnetic stir bar and a cooling jacket, under Ar atmosphere. The mixture was heated at 65 °C for 30 min until no more formation of H2 was observed and Analytical Chemistry, Vol. 78, No. 1, January 1, 2006

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the solution became pale green-yellow. At that moment, the solution was allowed to reach room temperature, and the corresponding phosphonium bromide salt 2-6 (6 mmol, 1 equiv) dissolved in anhydrous DMSO (5 mL) was added dropwise. A characteristic color shift to an intense red was produced, indicating the formation of the corresponding ylide. After 15 min, freshly distilled benzaldehyde (0.65 mL, 6 mmol, 1 equiv) was added and allowed to react for 2 h at RT under stirring until the starting material had disappeared according to TLC (hexane/ethyl acetate/ AcOH, 1:1:0.1). The crude of the reaction was acidified with 1 N HCl and extracted with ethyl ether. The organic phase was washed with H2O, dried with MgSO4, and filtered. The solvent was evaporated to dryness to obtain mixtures of the Z/E ω-phenylalkenoic acids that were subsequently esterified in MeOH with a few drops of concentrated H2SO4 at RT until the total disappearance of the starting material by TLC. The solvent was evaporated, and the residue was dissolved in ethyl ether and washed with saturated NaHCO3. The organic phase was dried with MgSO4, filtered, and evaporated. The crude mixture was purified by silica gel flash chromatography using hexane/ethyl ether as mobile phase to obtain the corresponding methyl esters as a Z/E mixture. Step 2: Synthesis of the ω-Phenylalkanoic Acids 1b-6b. The double bond of the ester (3.92 mmol) was reduced with H2 using Pd/C (10% Pd, 208.4 mg, 0.19 mmol Pd) as catalyst in MeOH (10 mL). The suspension was purged several times with vacuum/H2 cycles to remove the O2 present in the media and finally was kept under H2 at atmospheric pressure. The reaction mixture was stirred for ∼2 h at RT until the disappearance of the starting material was observed by TLC (hexane/ethyl ether, 1:1). The suspension was then purged again with vacuum/N2 cycles to eliminate the H2. The catalyst was removed by filtration, and the MeOH was evaporated to dryness to obtain the corresponding methyl ω-phenylalkanoates. The esters were hydrolyzed in THF and with 0.5 N NaOH at RT until the disappearance of the ester by TLC. The THF was removed under vacuum, and the aqueous solution was acidified with concentrated HCl and extracted with Et2O. The organic phase was dried with MgSO4, filtered, and evaporated to dryness to obtain the desired ω-phenylalkanoic acids 1b-6b. Preparation of the Sulfophenyl Carboxylates 5C5-, 6C6-, 7C7-, 9C9-, 12C12-, and 13C13-SPCs. Sulfonation of the phenyl carboxylic acids 1b-6b was performed following a similar procedure as described.35,36 The corresponding phenylcarboxylic acids 1b-6b (2.8 mmol, 1 equiv) were added to a round-bottom flask containing concentrated H2SO4 (1 mL, 18.22 mmol, 6.5 equiv) and equipped with a cooling jacket heated at 100 °C. The reaction mixture was stirred for 2 h and then slowly poured into H2O (80 mL). 5C5-, 6C6-, and 7C7-SPCs were isolated as calcium salts by washing the aqueous solution with Et2O (3 × 25 mL) and then neutralizing it with CaCO3 (6 g, 75 mmol). The solid formed was removed by filtration, and the aqueous solution was evaporated under reduced pressure to dryness to finally obtain the desired SPCs as calcium salts. 9C9-, 12C12-, and 13C13-SPCs were purified from the reaction mixture as sodium salts by extracting the

aqueous layer with ethyl acetate. The organic phase was dried with MgSO4, filtered, and evaporated to dryness to finally obtain a dark oil that was subsequently washed with a saturated solution of NaHCO3. The yellow precipitate obtained was filtered and washed with ethyl acetate to yield a white solid corresponding to the desired compound. Immunochemistry. General Methods and Instruments. The MALDI-TOF-MS (matrix-assisted laser desorption ionization timeof-flight mass spectrometer) used for analyzing the protein conjugates was a Perspective BioSpectrometry Workstation equipped with the software Voyager-DE-RP (version 4.03) developed by Perspective Biosystems Inc. (Framingham, MA) and Grams/386 (for Microsoft Windows, version 3.04, level III) developed by Galactic Industries Corporation (Salem, NH). The pH and the conductivity of all buffers and solutions were measured with a pH meter pH 540 GLP and a conductimeter LF 340, respectively (WTW, Weilheim, Germany). Polystyrene microtiter plates were purchased from Nunc (Maxisorp, Roskilde, DK). Washing steps were performed on a SLY96 PW microplate washer (SLT Labinstruments GmbH, Salzburg, Austria). Absorbances were read on a SpectramaxPlus (Molecular Devices, Sunnyvale, CA). The competitive curves were analyzed with a four-parameter logistic equation using the software SoftmaxPro v2.6 (Molecular Devices) and GraphPad Prism (GraphPad Sofware Inc., San Diego, CA). Unless otherwise indicated, data presented correspond to the average of at least two well replicates. Chemicals and Immunochemicals. Immunochemicals and the seasalts used to prepare artificial seawater were obtained from Sigma Chemical Co. (St. Louis, MO). The preparation of the protein conjugates and the antisera is described below. Most of the chemicals used for crossreactivity studies were acquired from Aldrich Chemical Co. (Milwaukee, WI). The technical mixture of LAS was kindly provided by PETRESA S.A. (San Roque, Ca´diz, Spain). The exact percentage weight of each LAS homologue was 0.5 units; As dilution > 1/1000; [ET] < 0.5 µg mL-1; [Ag] < 0.5 µg mL-1) were further investigated to test recognition of the technical mixture of LAS in solution. As a result of those experiments, several competitive assays were obtained in both formats. Table 2 shows those assays with IC50 values lower than 300 µg L-1. In contrast to the As raised against 13C13-SPC-KLH (As95-96) or to the mixture of immunogens (As97-98), no usable assays were obtained with the As raised against SFA-KLH (As93-94). On a direct ELISA format, only one As/ET combination (As98/7C7-SPC-HRP), using As generated by pseudoheterologous immunization procedures, provide Analytical Chemistry, Vol. 78, No. 1, January 1, 2006

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Table 2. Immunoassay Features of the Different Competitive ELISAsa immunogen

assay

Amax

Amin

IC50, µg L-1

slope

R2

13C13-SPC-KLH

As96/6C6-SPC-OVA As96/7C7-SPC-CONA As96/7C7-SPC-OVA As96/9C9-SPC-BSA As96/9C9-SPC-CONA As96/12C12-SPC-BSA As96/12C12-SPC-CONA As96/13C13-OVA As 98/7C7-SPC-HRPb As97/12C12-SPC-BSA As98/5C5-SPC-OVA As98/6C6-SPC-OVA As98/7C7-SPC-CONA As98/9C9-SPC-CONA As98/9C9-SPC-BSA

1.60 1.60 0.68 1.75 0.88 1.66 0.99 1.02 0.41 0.57 1.28 1.33 1.00 1.61 1.11

0.10 0.06 0.09 0.13 0.11 0.10 0.12 0.03 0.04 0.02 0.10 0.01 0.07 0.22 0.13

180.5 119.8 51.4 ( 18.4d 153.9 73.0 ( 29.2c 84.8 ( 28.4c 149.9 163.9 132.6 140.0 152.3 153.9 37.1 ( 3.2c 57.2 ( 9.0c 120.8

-0.8 -0.7 -0.4 -0.9 -0.5 -0.5 -0.8 -0.9 -0.57 -1.3 -1.1 -0.9 -0.7 -0.5 -0.8

0.97 0.99 0.97 0.81 0.99 0.92 0.96 0.97 0.64 0.98 0.88 0.97 0.92 0.99 0.97

SFA-KLH 13C13-SPC-KLH

a Only those assays showing reasonable parameters and IC -1 are shown. The values are extracted from the four50 values below 300 µg L parameter equation used to fit the standard curves. b Direct assay. All the other combinations shown are indirect assays. c The data presented correspond to the average of 5 calibration curves run in 5 different days.

Figure 4. Effect of the pH and the ionic strength on the Amax and IC50 of the direct (graphs A and B) and indirect (graphs C and D) ELISA assays developed for LAS analysis. The data are extracted from the four-parameter equation used to fit the standard curves.

acceptable features to continue with the evaluation. In contrast, several assays were obtained under an indirect ELISA configuration, using As obtained by both homologous and pseudoheterologous immunization strategies. However, repetitive experiments using the best assays obtained with each set of As demonstrated that As98/7C7-SPC-CONA was the most reproducible assay (see standard deviations for the IC50 values in Table 2). In both ELISA formats, the As giving the best assays were obtained through pseudoheterologous immunization methods. Moreover, hapten 7C7-SPC has always provided the assays with the best features, supporting that seven carbon units is a critical spacer length also under competitive configurations. Evaluation and Characterization of the ELISA Methods. Both direct (As98/7C7-SPC-HRP) and indirect (As98/7C7-SPCCONA) ELISAs were further investigated with the aim to improve performance and to characterize their behavior in media with different physicochemical parameters (pH, ionic strength, etc.). 78 Analytical Chemistry, Vol. 78, No. 1, January 1, 2006

Preincubating the analyte with the As, previous to the competitive step, for 15 and 60 min in direct and indirect assays, respectively, improved the detectability. Similarly, both direct and indirect formats appeared to perform much better in the absence or very low concentration of Tween 20 (