Broad-Specificity Immunoassays for Sulfonamide Detection

reactivity pattern of antibodies for the family of sulfon- amide drugs, a novel strategy based on the single-ring. (fragment-derived) hapten moieties ...
0 downloads 0 Views 195KB Size
Download PDF
Anal. Chem. 2006, 78, 1559-1567

Broad-Specificity Immunoassays for Sulfonamide Detection: Immunochemical Strategy for Generic Antibodies and Competitors Milan Franek,* Iva Diblikova, Ivo Cernoch, Maria Vass, and Karel Hruska

Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic

Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the crossreactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (onering) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The tworing 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 µg‚L-1 (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 µg‚L-1. This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site. Sulfonamides (SA) are antimicrobial agents widely used in veterinary medicine for the prophylaxis and therapy of infectious diseases, implying the risk of drug residues in food of animal origin. Therefore, an efficient system for the control of the residues of these veterinary drugs is required. In the Czech Republic, 28 remedies containing 6 sulfonamides (sulfachloropyridazine, sulfachloropyrazine, sulfadimethoxine, sulfadoxine, sulfadiazine, sulfamethazine) are registered for veterinary application to food producing animals. Other European countries, and also the United * Corresponding author. Fax: +420 5 41211229. E-mail: [email protected]. 10.1021/ac0514422 CCC: $33.50 Published on Web 02/02/2006

© 2006 American Chemical Society

States and Canada, use the same or a higher number of these drugs.1 In The Netherlands, at least nine sulfonamides are approved for veterinary use, five of which (sulfamethazine, sulfadiazine, sulfachlorpyridazine, sulfaquinoxaline, sulfamethoxazole) are approved for the medication of chicken.2 Sulfonamides are derivatives of sulfanilamide () p-aminobenzenesulfonamide), which are usually defined as N1- or N4substitued compounds depending on the substitution of the amido or aromatic amino group, respectively. Substitution at the aromatic amino group of a sulfonamide molecule results in N4-acetylated compounds that are metabolic products of the drugs. Substitutions at the amido group with heterocyclic aromatic nuclei results in N1 compounds that vary in degrees of antimicrobial activity.3 Figure 1 shows the chemical structures of sulfonamides differing in their N1 substituents. p-Aminobenzenesulfonamide represents a moiety common to all these compounds.The maximum residue limit (MRL) for edible tissues was determined to be 100 µg/kg for total sulfonamide content in the EU, Canada, and United States.4-6 For evaluation of the presence of antibiotics residues in food matrixes, monitoring programs have been introduced in which immunochemical screening methods may play one of the key roles. Traditional methods for sulfonamide residue analysis are microbiological tests and analytical methods such as thin-layer chromatography or high-performance liquid chromatography. Chemical methods are time-consuming and labor intensive, whereas microbiological control based on inhibition tests usually require 2-3 days for microbe growth or may be nonspecific or lack the necessary MRLs for desirable residue monitoring. Therefore, specific and sensitive methods are needed allowing screening of sulfonamides within a quick and simple test.1,7 Antibody-based assays represent an alternative to instrumental chemical methods. Immunoassays have addressed both environmental and food testing needs, quickly becoming the screening (1) Cliquet, P.; Cox, E.; Haasnoot, W.; Schachet, E.; Goddeeris, B. A. J. Agric. Food Chem. 2003, 51, 5835-5842. (2) Haasnot, W.; Bienenmann-Ploum, M.; Kohen, F. Anal. Chim. Acta 2003, 483, 171-180. (3) Haasnoot, W.; Cazemier, G.; Du Pre, J.; Kemmers-Voncken, A.; BienenmannPloum, M.; Verheijen, R. Food Agric. Immunol. 2000, 12, 15-30. (4) The European Agency for the Evaluation of Medicinal Products; Sulphonamides (2) Summary Report. EMEA/MRL/026/95, 1995. (5) EU Regulation 508/1999, L60 (9-3-1999); 1999; pp 16-52. (6) Food and Drug Regulation, Canada Gazette Part II, Table 3, Division 15, Part B, 1991, 125, 1478-1480. (7) Muldoon, M. T.; Font, I. A.; Beier, R. C.; Holtzapple, C. K.; Young, C. R.; Stanker, L. H. Food Agric. Immunol. 1999, 11, 117-134.

Analytical Chemistry, Vol. 78, No. 5, March 1, 2006 1559

Figure 1. Structures of sulfonamides used for dose response testing.

method of choice for many compounds.8 Analysis of samples by immunoassays often does not require extraction or cleanup steps, making these assays particularly suitable for screening evaluation.9 Due to high sample throughput, these methods can dramatically reduce the number of analyses required to characterize food samples for sulfonamide contamination.10 Until recently, most of the sulfonamide assay developments have been focused on antibodies raised against a single compound such as sulfamethazine (sulfadimidine), sulfamerazine, sulfapyridine, sulfathiazole, sulfadimethoxine, or sulfachlorpyridazine.10-22 In most cases, these antibodies have been produced by immunogens synthesized (8) Franek, M.; Hruska, K. Vet Med. 2005, 50, 1-10. (9) Diblikova, I.; Cooper, K. M.; Kennedy, G.; Franek, M. Anal. Chim. Acta 2005, 540, 285-292. (10) Franek, M.; Kolar, V.; Deng, A. P.; Crooks, S. Food Agric. Immunol. 1999, 11, 339-349.

1560

Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

by linking the drug to a protein carrier via the aromatic amino group.3 Thus, the N1 group is situated at a site opposite to the spacer linkage and is maximally exposed to the antibody for recognition. Surprisingly, when the common aminobenzene(11) Lee, N.; Holtzapple, C. K.; Muldoon, M. T.; Deshpande, S. S.; Stanker, L. H. Food Agric. Immunol. 2001, 13, 5-17. (12) Crabbe, P.; Haasnoot, W.; Kohen, F.; Salden, M.; Van Peteghem, C. Analyst 1999, 124, 1569-1575. (13) Garden, S. W.; Sporns, P. J. Agric. Food Chem. 1994, 42, 379-1391. (14) Ko, E.; Song, H.; Park, J. H. J. Vet. Med. Sci. 2000, 62, 1121-1123. (15) Muldoon, M. T.; Buckley, S. A.; Rocco, R. M.; Deshpande, S. S.; Stanker, L. H. Abstr. Pap. Am. Chem. S 1996, 212, 84-AGRO. (16) Muldoon, M. T.; Font, I. A.; Young, C. R.; Stanker, L. H. Abstr. Pap. Am. Chem. S. 1996, 211, 97-AGRO. (17) Muldoon, M. T.; Buckley, S. A.; Deshpande, S. S.; Holtzapple, C. K.; Beier, R. C.; Stanker, L. H. J. Agric. Food Chem. 2000, 48, 545-550. (18) Spinks, C. A.; Schut, C. G.; Wyatt, G. M.; Morgan, M. R. A. Food Addit. Contam. 2001, 18, 11-18.

sulfonamide moiety was situated at the immunodominant position by linkage through the N1 group, antibodies with a broad crossreactivity pattern were not formed. Haasnoot et al.3,23 and PastorNavaro et al.24 used various sulfonamide derivatives with the common moiety remote to the protein surface for immunization of animals in an attempt to raise generic antibodies. Their polyclonal and monoclonal antibodies were surprisingly specific, and only a limited number of sulfonamide species could be detected in ELISA. Spinks et al.25 proposed a strategy based on the heterologous combination of antibodies against sulfachloropyridazine and coating conjugates being modified at the N1 position. Although this reagent combination proved to have specificity, recognizing 15 out of the 21 drugs tested, the assay response to most of the sulfonamide species was weak and therefore not applicable in real screening analysis. Recently, recombinant antibodies with a broad-specificity pattern for the class of sulfonamide compounds have been prepared by Korpima¨ki et al.26-29 Mutagenesis and phage display selection26 were employed by these authors to manipulate analytical properties of the monoclonal antibody produced by Haasnoot et al.23 The use of protein engineering in the following development phases resulted in antibody mutants with broad selectivity, capable of simultaneous detection of 10-18 sulfonamides at the regulatory levels.27-29 From our previous study, it appeared that the cross-reactivity pattern for sulfonylurea herbicides was considerably enlarged when the s-triazine moiety, common to these herbicides, was linked to a carrier protein. In this case, a one-ring s-triazine moiety representing a part of the sulfonylurea molecule was employed as a fragment-derived hapten evoking a generic immune response.30 The produced monoclonal antibody exhibited a sensitive response toward eight related sulfonylurea species having a crossreactivity pattern in the range of 21-167%. The results prompted us to apply this approach to a broad family of sulfonamide drugs, which have an aromatic one-ring moiety common to them all. The unique chemical pattern of the sulfonamide species offered us a suitable model system to study the general validity of the above concept based on the use of the fragment-derived hapten approach. The aim of this study was to particularly explore the capability of the p-aminobenzenesulfonamide moiety attached to a carrier protein through the N1 position to evoke a generic (19) Thomson, C. A.; Sporns, P. J. Food Sci. 1995, 60, 872-879. (20) Thomson, C. A.; Sporns, P. J. Food Sci. 1995, 60, 409-415. (21) Cliquet, P.; Cox, E.; Haasnoot, W.; Schachet, E.; Goddeeris, B. A. Anal. Chim. Acta 2003, 494, 21-28. (22) Kohen, F.; Gayer, B.; Zaltsman, Y. A.; O’Keeffe, M. Food Agric. Immunol. 2000, 12, 193-201. (23) Haasnoot, W.; Du Pre, J.; Cazemier, G.; KemmersVoncken, A.; Verheijen, R.; Jansen, B. J. M. Food Agric. Immunol. 2000, 12, 127-138. (24) Pastor-Navarro, N.; Garcia-Bover, C.; Maquieira, A.; Puchades, R. Anal. Bioanal. Chem. 2004, 379, 1088-1099. (25) Spinks, C. A.; Wyatt, G. M.; Everest, S.; Jackman, R.; Morgan, M. R. A. J. Sci. Food Agric. 2002, 82, 428-434. (26) Korpima¨ki, T.; Rosenberg, J.; Virtanen, P.; Karskela, T.; Lamminma¨ki, U.; Tuomola, M.; Vehnia¨inen, M.; Saviranta, P. J. Agric. Food Chem. 2002, 50, 4194-4201. (27) Korpima¨ki, T.; Rosenberg, J.; Virtanen, P.; Lamminma¨ki, U.; Tuomola, M.; Saviranta, P. Protein Eng. 2003, 16, 1, 37-46. (28) Korpima¨ki, T.; Brockmann, E. Ch.; Kuronen, O.; Saraste, M.; Lamminma¨ki, U.; Tuomola, M. J. Agric. Food Chem. 2004, 52, 40-47. (29) Korpima¨ki, T.; Hagren, V.; Brockmann, E. Ch.; Tuomola, M. Anal. Chem. 2004, 76, 3091-3098. (30) Kolar, V.; Deng, A.; Franek, M. Food Agric. Immunol. 2002, 14, 41-105.

response of interest. The evaluation of the peroxidase tracers and coating conjugates based on sulfonamide-derived hapten fragments in heterologous assay systems was an additional objective of this work. EXPERIMENTAL SECTION Reagents and Chemicals. 2-(4-Aminobenzensulfonylamino)ethanoic acid, 4-(4-aminobenzensulfonylamino)butanoic acid, 6-(4aminobenzensulfonylamino)hexanoic acid, 4-(4-aminobenzensulfonylamino)benzoic acid, and 6-(4-aminobenzensulfonylamino)pyridine-3-carboxylic acid were synthesized at Biosfor (J. Socha; Na Labisti 533 53 009, Pardubice, Czech Republic). Bovine serum albumin (BSA), thyroglobulin (TG), ovalbumin (OV), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), horseradish peroxidase (HRP), swine immunoglobulin against rabbit immunoglobulin-HRP (SwAR), Freund’s Complete Adjuvant (FCA), Freund’s Incomplete Adjuvant, Sephadex G-25, p-aminobenzoic acid, sulfamethazine, sulfamerazine, sulfadimethoxine, sulfacetamide, sulfamethoxazole, sulfachloropyridazine, sulfamethoxypyridazine, sulfapyridine, sulfathiazole, sulfamethizole, sulfaguanidine, and sulfisoxazole were obtained from Sigma Aldrich Chemie (Steinheim, Germany). 3,3′,5,5′-Tetramethylbenzidine was purchased from Serva (Heidelberg, Germany). Sulfadiazine, sulfamethoxydiazine, sulfanilamide, sulfachloropyrazine, sulfaquinoxaline, sulfadoxine, and sulfaphenazole were donated by K. Frgalova (Institute for State Control of Veterinary Biologicals and Medicaments, Brno, Czech Republic); N4-acetyl sulfadiazine and N4 acetyl sulfamethazine were received from CH. T. Elliott (Veterinary Sciences Division, Department of Agriculture and Rural Development, Belfast, Nothern Ireland, U.K.). Instrumentation. Maxisorp microtiter plates were purchased from Nunc (Roskilde, Denmark) and Vivapore 5 mL concentrators from Vivascience (Hannover, Germany). Autostrip washer EL 50 (Bio-Tec Instruments, Inc.) was used for washing the microtiter plates. Ultramicroplate reader EL 808 with software KC4 v3.1 (BioTec Instruments, Inc.) and Origin v7.5 (OriginLab Corp.) were used for the absorbance measurement in plates and the processing of ELISA results. Buffers and Solutions. The following solutions were used: (1) coating buffer, prepared as 50 mmol‚L-1 carbonate buffer (pH 9.6); (2) assay buffer (phosphate-buffered saline, PBS), 10 mmol‚L-1 phosphate buffer with 145 mmol‚L-1 NaCl (pH 7.2); (3) assay buffer-BSA, 10 mmol‚L-1 phosphate buffer with 145 mmol‚L-1 NaCl (pH 7.2), 5 g‚L-1 BSA (blocking agent); (4) wash buffer: PBS buffer with 0.1% (v/v) Tween 20 (PBST20), (5) acetate (substrate) buffer, 0.1 mol‚L-1 sodium acetate adjusted to pH 5.5 by addition of 1 mol‚L-1citric acid; (6) dialysis solution, 0.1 mol‚L-1 (NH4)2CO3 in water; (7) 0.13 mol‚L-1 NaHCO3 in water; (8) TMB solution, 10 mg‚mL-1 TMB in DMSO; (9) substrate solution, prepared by addition of 1 mL of acetate buffer, 200 µL of TMB solution, and 20 µL of 6% H2O2 to 20 mL water; (10) 2 mol‚L-1 H2SO4 as the stopping reagent. Standards. Stock solutions were prepared by adding 1 mg of sulfonamide to 10 mL of methanol (100 mg‚L-1). For the calibration series and for testing inhibition reactions, series of concentrations were prepared in the range of 0.5-10 000 µg‚L-1, using individual sulfonamides. An equimolar mixture consisting of 19 sulfonamides was prepared from aliquots of each stock Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

1561

Figure 2. Structures of hapten derivatives for the conjugation with proteins: (I) 2-(4-aminobenzensulfonylamino)ethanoic acid, (II) 4-(4aminobenzensulfonylamino)butanoic acid, (III) 6-(4-aminobenzensulfonylamino)hexanoic acid, (IV) 2-(4-acetaminobenzensulfonylamino)ethanoic acid, (V) (4-(4-aminobenzensulfonylamino)benzoic acid, (VI) 6-(4-aminobenzensulfonylamino)pyridine-3-carboxylic acid, and (VII) p-aminobenzoic acid.

solution. The 1 mL volumes of individual stock solutions were mixed followed by addition 1 mL of methanol to obtain a final volume of 20 mL. The concentration of the standard sulfonamide mixture was 95 mg‚L-1. Molecular Modeling. Modeling studies were carried out using the “systematic search” procedure implemented in the CICADA program (the single coordinate during SCD method). The energy calculations were performed using molecular mechanics, MM3 force field. The global minimum found using the CICADA approach was optimized using the Gaussian 98 program. The DFT B3LYP method was used for energy calculation (basis set at 6-316 (d,p)). The atomic charges (Mulliken charges) and electrostatic potential of the molecules were calculated using the Gaussian 98 with the same basis set. Synthesis of Sulfonamide Conjugates. The hapten derivatives (Figure 2) were conjugated to OV, BSA, and TG via the end carboxylic group using the N-hydroxysuccinimide ester method as follows: Hapten derivative (0.32 mmol), NHS (0.32 mmol), and DCC (0.32 mmol) were each dissolved in DMF (200 µL), and the mixture was stirred at 4°C overnight. The activated haptens were centrifuged for 10 min at 700g, and the of supernatant was added dropwise under continuous stirring to 100 mg of OV, BSA, and TG in 0.13 mol‚L-1 NaHCO3 (2 mL) to obtain the final molar ratios 1:50, 1:100, and 1:200 (protein/activated hapten), respectively. The mixtures were stirred at 4°C overnight to complete conjugation. The protein conjugates were centrifuged for 10 min at 1120g, dialyzed against 0.1 mol‚L-1 (NH4)2CO3, and, after concentration using Vivapore concentrators, purified on Sephadex G-25. Frac1562

Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

tions containing the conjugate were collected, lyophilized, and stored at 4°C. Hapten-HRP Tracers. The hapten derivatives (with end carboxylic groups) (5 µmol), NHS (5 µmol, 0.58 mg), and DCC (5 µmol, 1.03 mg) were dissolved in DMF (60 µL). The mixture was gently stirred at room temperature for 18 h. The activated hapten was centrifuged at 490g for 10 min, and 27.6 µL of the supernatant was added dropwise while stirring to 3.4 mg of HRP in 0.13 mol‚L-1 NaHCO3 (300 µl) in order to obtain molar ratio 10:1 (hapten/HRP). The mixture was stirred at room temperature for 5 h, and the formed tracer was purified using Sephadex G-25 with 0.01 mol‚L-1 NaHCO3 as the eluent. The tracer conjugate obtained was diluted with the same volume of glycerol and stored at -20°C until use. Immunization. Immunization of New Zealand White and Californian White rabbits was carried out by intradermal administration of the immunogens after emulsification in Freund’s Adjuvant. Immunogens (0.75 µg) were dissolved in 0.75 mL of PBS and emulsified in 0.75 mL of Freund’s Complete Adjuvant. The emulsion (one immunization dose) was injected intradermally into 15 sites on the back region of the animal in 100-µL aliquots. The repeated injections using Freund’s Incomplete Adjuvant followed in 30-day intervals. Ten to twelve days after the fifth booster injection, blood samples for the preparation of antisera were collected, and the serum was stored at -20 °C. Direct ELISA. Microtiter plates were coated at 4°C overnight with 200 µL well of diluted antiserum in coating buffer. The coated plates were washed three times with wash buffer to remove unbound components. A 100-µL aliquot of standard or sample in assay buffer and then 100 µL of HRP-tracer in assay buffer with BSA were added to the wells. The plates were shaken on a microtiter plate shaker for 1 min and then incubated 1 h at 4 °C. After washing three times, 200 µL of enzyme substrate and chromogen was added, followed by 15-min incubation at room temperature. The enzymatic reaction was stopped by addition of 2 mol‚L-1 H2SO4 (100 µL/well). The absorbances were measured at 450 nm. Indirect ELISA. Immunogens were prepared in coating buffer (0.2 g‚L-1) and 200 µL was added per well, as the solid-phase immobilized agents. Microtiter plates were incubated overnight at 4°C and thereafter washed three times with wash buffer. Addition of 100 µL of standard (or sample) and 100 µL of antiserum, both in assay buffer, then followed. Plates were then incubated for 1 h at 4°C and washed, and 100 µL of SwAR in PBST20 was added into the wells. The plates were further incubated (1 h at 4°C) and developed as described previously. Milk Samples. Antibiotic-free milk from two dairy cows was taken into sample bottles. The collected samples of the raw milk were mixed, cooled, and transported to the laboratory. The milk was centrifuged (1100g, 15 min, 4°C), the upper fat layer was mechanically removed, and the defatted milk was used for the preparation of the calibration series as described for the assay buffer. Skimmed milk was purchased from a milk shop and used for the preparation of matrix-matched standards by the same procedure used for the raw milk. The 100-µL samples of milk were taken for the ELISA analysis.

RESULTS Hapten Design and Competitors. Our intended approach to hapten design was the generation of sulfonamide group-specific antibodies and was based on the following assumptions. First, single-ring hapten moieties (Figure 2, structures SA1-) were designed for conjugation with carrier proteins to raise dominant specificity toward the common p-aminobenzenesulfonamide group. Since this moiety represents only a part of the drug structure, it is referred to as a fragment-derived hapten moiety. The sulfonamide fragment moiety was expected to be an antigenic entity evoking a generic response of our interest. This premise was supported by a fundamental notion that the binding site raised against the antigenic determinant is formed according to principle of immunological complementarity. The classical concept of complementarity is practically identical to the idea of binding specificity, since the regions complementary in shape are understood as being specific.31 In our case, binding interaction can be expected between the binding site and the p-aminobenzenesulfonamide moiety of the sulfonamide molecule. N1 substituents that confer a unique identity to the moieties do not participate in binding due to the absence of the steric and charge complementarity (Figure 1). In other words, acceptance of the existence of the single-ring binding model eliminates the possibility of a binding interaction of the N1 moiety with the antibody binding site. Second, the SA1- haptens must be attached to proteins via spacer substituents introduced into the hapten at the position opposite to the aromatic amino group. Thus, the sulfonamide hapten moiety will enter the antibody-binding cavity of the aromatic amino group, whereas the N1 bulky aromatic group will remain in a loose-fit area or is completely outside the binding site. Interaction with the binding site will be implemented particularly at sites of the hapten moiety, which are removed from the spacer substituent (i.e., in areas with the highest degree of structural complementarity), whereas the existence of the loose-fit area eliminates the possibility of a specific interaction between the N1 substituent and the antibody. Third, we assume that the presence of the acetyl group at the N4 immunodominant position prevents it from the binding interaction among metabolized sulfonamides and the antibody binding site; thus, minimum metabolite recognition can be expected. Fourth, the SA1- carboxylic derivatives having a different length of the spacer substituent were used for attachment to carrier proteins to raise antibodies of different selectivity to sulfonamides. This spacer strategy was based on the notion that an immunogen with a short chemical bridge between the hapten and a protein may generate antibodies with a low ability to discriminate the structural changes at the N-1 position (generic antibodies) whereas a long spacer chain can elicit a different degree of binding specificity.32 Five, a two-ring structure, SA2(Figure 2), selected on the basis of molecular modeling7 was used to verify the ability of this hapten determinant to generate a broad class-specificity response. We intended also to employ this structure in combination with the SA- haptens to improve assay characteristics in heterologous ELISA formats. The SA3- carboxylic derivative, based on the N1 pyridine moiety, mimicked the sulfonamide heterocyclic pattern more authentically than the SA2(31) Franek, M. J. Steroid Biochem. Mol. Biol. 1987, 28, 95-108. (32) Franek, M.; Zeravik, J.; Eremin, S. A.; Yakovleva, J.; Badea, M.; Danet, A.; Nistor, C.; Ocio, N.; Emneus, J. Fresenius J. Anal. Chem. 2001, 371, 456466.

Table 1. Hapten Density in BSA Conjugates conjugate (immunogen)

reaction (mol/mol)

ratio hapten density (mol/mol)

SA1-CH2-BSA SA1-(CH2)3-BSA SA1-(CH2)5-BSA1 SA1-(CH2)5-BSA2 SA1-(CH2)5-BSA3 SA1-(CH2)5-BSA4 SA2BSA ABBSA AcetSA-CH2-BSA

1:100 1:100 1:10 1:50 1:100 1:200 1:100 1:100 1:100

14 ( 1 11 ( 1 4(1 20 ( 2 47 ( 1 53 ( 1 34 ( 1 15 ( 1 9(2

structure. Finally, the influence of the central region of the sulfonamide molecule and the SO2NH bridge moiety, on the character of the immune response was studied using conjugated p-aminobenzoic acid. Absence of the central sulfonamide part in this molecule (Figure 2, structure AB-) allowed us to assess the importance of this subdeterminant for the production of antibodies against sulfonamides. Conjugates, Antibodies, and Assay Optimization. The conjugation reactions were carried out using the hapten derivatives, carrier proteins, and HRP. Eighteen various SA-, three AB-, three acetyl-SA- protein conjugates and seven HRP tracers with SA- and AB- moieties were prepared by the conventional Nhydroxysuccinimide/carbodiimide method. The degree of the hapten substitution in albumin conjugates was determined by MALDI-TOF MS analysis (Table 1). The data presented in Table 1 demonstrate that reaction molar ratios between haptens and carrier proteins used for the conjugation reactions reflected hapten densities well in the final product. The conjugates SA1-(CH2)5BSA1-BSA4 prepared at the reaction ratios 1:10, 1:50, 1:100, and 1:200 contained 4, 20, 47, and 53 hapten groups per protein molecule, respectively. The hapten rates incorporated into ovalbumin, thyroglobulin, and HRP carriers could not been assessed due to the low reproducibility and therefore difficult interpretability of MS spectra. Immunogens based on hapten structures in Figure 2 were used for immunization of 43 rabbits. The presence of antibodies in sera was monitored by hapten homologous and heterogeneous assay combinations using coating conjugates based on the immunizing hapten structures. Sera having antibody titer higher than 1:1000 in indirect ELISA were tested for their competitive capability using the equimolar mixture of 19 sulfonamides. Only 3 combinations of the coating conjugates and antibodies of the 106 tested showed a competitive response to the sulfonamides and strong binding in reagent dilutions 1:5000 or higher. As apparent from Table 2, the coating dilution for the immobilized conjugates was 1:5000 and 1:10000, and the corresponding antibody dilutions were 1:50000 and 1:40000 in the optimized indirect assays. Antisera against acet-SA1-CH2-protein conjugates exhibited high antibody titers (not included in Table 2); however, their further characterization could not be carried out due to the unavailability of discontinued standards from supplier (Sigma Aldrich). In other experiments, the effect on the degree of hapten density in the conjugate on dose response curves used for well coating was investigated. The curves were determined according to the standard indirect ELISA protocol using the same coating concentration for conjugates with different hapten incorporation. Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

1563

Table 2. Optimized Reagent Dilutions for Various Reagent Combinations

a

combination no.

coating conjugatea

Indirect ELISA dilutionb

antibody (rabbit)

dilution

1 2 3

SA1-CH2-TG SA1-CH2-TG SA1-(CH2)5-TG

1:5000 1:10000 1:10000

anti SA1-CH2-BSA (No. 2) anti SA1-CH2-OV (No. 6) anti SA2-OV (No. 43)

1:50000 1:40000 1:50000

combination no.

coating antibody (rabbit)

4 5 6 7 8c

anti SA1-CH2-BSA (No. 2) anti SA1-CH2-OV (No. 6) anti SA2-TG (No. 39) anti SA2-OV (No. 43) anti SA2-OV (No. 43)

Direct ELISA dilution 1:50000 1:25000 1:20000 1:20000 1:15000

tracer

dilution

SA1-(CH2)5-HRP SA1-(CH2)5-HRP SA1-(CH2)5-HRP SA1-(CH2)5-HRP SA1-(CH2)5-HRP

1:100000 1:50000 1:150000 1:150000 1:50000

The respective hapten structures are depicted in Figure 2. b The concentration before dilution was 0.2 g‚L-1. c Skimmed milk.

Figure 3. Effect of hapten density of the coating SA1-(CH2)5-BSA conjugate on the dose response curves using antibody 43. The coating concentration of the conjugate was 10 ng.mL-1; the antibody dilution was 1:100000.

The results of this measurement are shown in Figure 3. The dose responses for curves using the less substituted SA1-(CH2)5-BSA conjugates were at least 1 order of magnitude lower than when using conjugates with 1:100 and 1:200 degrees of substitution. The comparison of the curve sensitivity in Figure 3 was consistent with maximum assay sensitivity, because differences in absorbance values at zero concentration of the standard were small, ranging between 0.95 and 1.25 absorbance units. The antibodies were further examined for binding interactions using HRP tracers with different hapten moieties and spacer lengths. As shown in Table 2, the coating dilution for the best antibodies were 1:15000 to 1:50000 whereas the assay dilutions for SA1-(CH2)5-HRP tracer ranged from 1:50000 to 1:150000. The results of inhibition testing obtained from 42 direct assays based on various combinations of seven antibodies and six tracers are summarized in Table 3. The IC50 values assessed for the sulfonamide mixture ranged within 1.5 and 133 µg‚L-1. The assays using a tracer bearing a one-ring moiety with a long spacer (SA1-(CH2)5-) generated the most sensitive response in all antibodies tested. However, HRP tracers based on the two-ring moieties (SA2- and SA3-) exhibited responses approaching in some cases the sensitivity obtained by SA1-(CH2)5-HRP conjugate. One-ring SA1-CH2-, 1564 Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

SA1-(CH2)3- haptens, having shorter spacer arms, exhibited good sensitivity only in some reagent combinations whereas a low binding was found for additional antibodies combined with these tracers. Based on these data, the reagent combinations 4-7 (Table 2) were chosen for further characterization. SA1-(CH2)5-HRP was employed as a common competitor for all ELISA systems tested. Dose Response to Individual Sulfonamides. To assess group specificity in terms of individual sulfonamide responses, 19 sulfonamides were tested for their inhibition efficiencies. The results of competitive reactions for seven combinations of reagents are summarized in Tables 4 and 5. The concentration of sulfonamide inhibiting 20, 50, and 80% of tracer binding in the assay was determined to indicate the dose responses throughout all the calibration range. The IC20 value represents the concentration at which the respective sulfonamide can be reliably detected by the assay, whereas the IC50 values are the detection regions with the highest assay sensitivity. Antibodies 2, 6, and 43 were incorporated into both indirect and direct assay formats whereas antibody 39 was employed only in combination with the HRP tracer since binding interaction of this antibody with coating conjugates was poor. The results in Table 4 show that the indirect ELISAs were capable of detecting 8-17 and 2-10 individual sulfonamides at the 100 ppb level with the sensitivity corresponding to the IC20 and IC50 values, respectively. A relatively broad spectrum of sulfonamide responses was observed for antibody 43, but the response to commonly used SMZ was in this case not sufficient with respect to screening needs. Use of HRP tracers improved sensitivity toward SMZ and some other sulfonamides as shown in Table 5. A superior generic pattern was provided by reagent combination 6 and 7 working with antibodies 39 and 43, respectively, and tracer SA1-(CH2)5-HRP. These particular ELISAs detected at least 17 and 18 sulfonamides with the sensitivity of the IC20 and IC50 values, respectively, corresponding to the regulatory level. In reagent combination 7, a 50% binding decline was achieved by SMZ at 105 µg‚L-1 and the assay detected this drug at 3.7 µg‚L-1 in assay buffer. The antibodies exhibited a negligible assay interference (cross-reactivity less than 0.1%) with N4-acetylsulfamethazine, N4-acetylsulfadiazine, tetracycline, oxytetracycline, peniciline, and chloramfenicol. The antibodies did not react with p-aminobenzoic acid, which resembles the common part of sulfonamide drugs. Therefore, the ELISAs based on these

Table 3. IC50 Values (µg‚L-1) for the Sulfonamide Mixture Using Various Reagent Combination hapten-HRPa

a

antibody no.a

SA1-CH2-

SA1-(CH2)3-

SA1-(CH2)5-

SA2-

SA3-

AB-

anti-SA1-CH2-BSA (No. 2) anti-SA1-CH2-OV (No. 6) anti-SA2-OV (No. 43) anti-SA2-TG (No. 39) anti-SA1-CH2-BSA (No. 22) anti-SA1-CH2-TG (No. 24) anti-SA1-CH2-OV (No. 26)

5.8 1.5 lb nb 3.0 lb lb

lbb 17.3 15.5 133.0 lb lb lb

4.4 1.7 5.4 4.1 6.3 3.4 5.8

lb lb 4.3 9.8 lb lb 8.1

2.7 5.3 23.9 8.4 lb lb lb

nbc nb lb nb nb nb nb

Structures of the haptens are depicted in Figure 2. b lb, low binding. c nb, no binding.

Table 4. Inhibition Characteristic (IC20-80) for Individual Sulfonamides in Optimized Indirect ELISA Systemsa IC (µg‚L-1)

aSMZ

SMR

SDZ

SMDZ SAN SCR

SQ

SDM

SD

SAM

SMX

SCP

SMP

SP

STZ

SEZ

SG

SIX

SPZ

2)b

IC20 IC50 IC80

959 115 261 4393 534 2888 >10K 2480 >10K

73.6 1461 >10K

9.9 53.1 284

Reagent Combination No. 1 (Anti No. 5.8 258 34.4 58.0 1624 179 1.0 210 2298 830 1244 >10K 3266 152 7524 >10K >10K >10K >10K >10K >10K

0.8 9.4 110

10.2 183 227 159 341 4966 3764 867 >10K >10K >10K 4721

2634 787 >10K 4343 >10K >10K

IC20 IC50 IC80

98.7 60.0 64.6 877 452 276 >10K 3404 1181

1.8 19.0 198

10.3 79.9 618

Reagent Combination No. 2 (Anti No. 6) 15.6 15.9 1.4 31.9 127 0.6 0.1 255 83.3 8.5 736 591 4.9 0.8 4184 437 53.2 >10K 2752 41.3 6.0

0.05 0.4 3.5

0.6 3.5 20.8

0.9 12.3 175

0.5 6.5 80.1

25.5 700 >5K

643 37.7 9952 252 >10K 1681

IC20 IC50 IC80

21.3 247 2873

1.2 11.1 99

Reagent Combination No. 3 (Anti No. 43) 23.6 34.5 2.4 0.4 23.5 459 1.0 22.4 237 302 28.8 4.7 155 1581 8.6 131 2387 2661 348 54.5 1028 5440 77.4 765

2.1 13.7 88.7

0.2 2.2 22.6

3.0 39.4 519

1.1 4.8 20.6

1.8 116 206 749 >10K 4824

a

4.9 14.0 100 132 2059 1240

1.4 21.4 323

Abbreviations are defined in Figure 1. b Dilution of reagents is given in Table 2.

Table 5. Inhibition Characteristic (IC20-80) for Individual Sulfonamides in Optimized Direct ELISA Systemsa IC (µg‚L-1)

aSMZ

SMR

SDZ

SMDZ

SAN

SCR

SQ

SDM

SD

SAM

SMX

SCP

SMP

SP

STZ

SEZ

SG

SIX

SPZ

0.3 3.8 56.3

0.1 0.6 3.0

0.1 0.7 5.4

0.4 12.5 402

7.3 70.3 702

19.8 1201 >10K

2966 >10 K >10K

586 3098 >10K

0.02 0.1 0.3

0.3 4.1 122

2.3 18.2 145

1.8 16.2 83

159 3208 >10K

266 3333 >10K

10.3 223 7513

2)b

Reagent Combination No. 4 (Anti No. 0.2 5.0 482 787 5.2 4.0 171 1731 2367 255 79.3 5000 6331 7168 >10K

IC20 IC50 IC80

25.1 582 >10 K

1.0 16.1 264

1.2 18.2 418.5

0.2 6.7 513

8.4 43.5 235

13.5 237 4173

IC20 IC50 IC80

155 1426 >10K

11 443.8 >10K

14.9 405 1523

1.3 12.7 122

7.3 63.0 544

Reagent Combination No. 5 (Anti No. 6) 110.3 25.7 0.6 151 103.0 0.2 0.05 2871 140 8.9 897 1281 1.1 0.2 >10K 799 185 5333 >10K 17.0 1.6

IC20 IC50 IC80

20.6 311 4987

2.7 36.2 491

2.0 19.5 187

0.2 2.7 37.2

2.1 19.0 181

4.5 27.1 190

Reagent Combination No. 6 (Anti No. 39) 0.2 0.7 116.4 51 0.1 2.1 1.4 6.1 426 452 0.4 10.7 7.8 52.4 1598 5147 4.7 54.9

0.1 0.6 4.0

0.1 0.4 3.0

2.6 13.7 74.3

2.8 8.8 29.0

152 731 3520

10.5 95.6 701

14.9 49.5 151

IC20 IC50 IC80

3.7 105 3008

3.4 38.0 1093

4.3 29.6 205

0.2 2.6 29.5

8.4 57.0 388

12.0 79.1 523

Reagent Combination No. 7 (Anti No. 43) 0.3 1.4 20.8 113.0 0.1 3.7 2.1 10.4 104 758 1.39 20.3 13.3 113 1150 5670 18.4 118

0.2 1.3 7.8

0.1 0.4 2.4

1.3 8.2 54.7

12.0 21.4 193

6.2 59.4 575

82.5 390 1840

12.1 28.4 66.7

IC20 IC50 IC80

8.0 56.4 444.3

2.5 11.8 55.8

3.1 12.2 48.2

0.4 2.1 12.4

Reagent Combination No. 8, Skimmed Raw Milk (Anti No. 43) 5.5 4.0 0.5 0.7 3.7 65.2 0.4 1.9 0.4 26.0 17.1 2.7 3.2 23.8 294.1 1.8 9.7 2.4 125.7 74.4 16.5 14.6 157 1326 11.1 56.1 15.8

0.2 0.9 6.5

0.4 3.7 44.2

4.5 11.1 32.3

2.6 26.7 279.1

36.8 185.5 969

20.9 48.7 115.0

a

Abreviations are defined in Figure 1. b Dilution of reagents is given in Table 2.

antibodies have been considered to be group-specific assays detecting only parent sulfonamides. Data presented in Table 5 show that the difference in assay sensitivity observed for tested sulfonamide targets varied considerably, the least being in SMZ and SD and the highest in SP. Figure 4 shows calibration curves for three selected analytes differing in their dose responses. From these curves, the different

sensitivities toward SMZ, the sulfonamide mixture, and SP are evident. As expected, the curve obtained for the equimolar sulfonamide mixture was more sensitive than that belonging to SMZ and less sensitive than the most sensitive curve for SP. Responses in Milk. Individual sulfonamides were tested for their responses in undiluted skimmed milk originating from dairy cows and consumer sources. The results obtained from the raw Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

1565

Figure 4. Dose response curves for three sulfonamide analytes. ELISA was based on the antibody 43 and SA1-(CH2)5-HRP reagent combination. The coating was carried out at an antibody dilution of 1:20000 and the tracer dilution was 1:150000.

skimmed milk are summarized in Table 5. The assay, capable of detecting 19 sulfonamides, has a stronger inhibition activity in 12 species in the milk matrix than in assay buffer. A complex milk matrix effect and perhaps changes in sulfonamide solubility may explain this observation. Similar trends in results were obtained from the additional analysis of four skimmed milk products purchased from a milk shop, demonstrating the possible benefit of using the assay for multiple detecting of sulfonamides in undiluted samples of milk. DISCUSSION Several hapten designs have been used to manipulate antibody properties during the development of the broad-specificity immunoassays for sulfonamides, but the use of the immunogens based on one-ring (fragment-derived sulfonamide) haptens has up until now not been explored. The generic character of immunogens used in this work was demonstrated by a cross-reactivity pattern in the produced antibodies. However, interpretation of the inhibition data in structural terms is complicated due to the variability in the respective values determined for each individual antibody. The IC50 (SQ) established for antibody 2 was 4.0 µg‚L-1, while the same level of inhibition for antibody 6 (raised against the same SA1-CH2-hapten) was achieved at 140 µg‚L-1 in direct ELISA (Table 5). Different properties of antibodies can also be demonstrated by the remarkably different IC50 values established for SMX in a direct ELISA format (255 versus 0.4-1.4 µg‚L-1) (Table 5). The pattern of great inhibition differences was also observed for other tested sulfonamides (SMZ, SMR, SCR, SDM, SCP, SPZ). In general, drugs with a ring system free of substituents tended to have IC50 values of 1-2 orders of magnitude lower than the sulfonamides bearing one or two groups at the heterocyclic ring (SMZ, SIX, SD, SPZ). SDM, bearing two methoxy groups at the heterocyclic ring, evoked a considerably stronger response in antibodies than SMZ, which has two methyls at the heterocyclic ring with different nitrogen positions (Tables 4 and 5). A significant difference in charge distribution between R groups in these sulfonamide ligands could explain this observation (unpublished results). The smallest inhibition strength (low assay sensitivity) was observed in SAM and SG containing N1-alkyl substituents. Despite different responses to sulfonamide analytes in individual 1566 Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

Figure 5. Partial atomic charges calculated for p-aminoacetic acid hapten [A] and sulfacetamide with a SO2-NH central grouping [B]. Charges were calculated on B3LYP/6-31 (d,p) level of theory.

antibodies, the data obtained are not contradictory to our initial notion that the N1 moieties remain in a loose area or outside the binding site, whereas a high-affinity interaction takes place through the common p-aminobenzenesulfonamide moiety inside the cavity of the binding site. Although this concept emphasizes steric aspects of the sulfonamide-antibody interaction, the possible influence of the charge/protonation contributions of the sulfonamide competitors on the binding affinity should not be overlooked. Various charge distributions across the whole sulfonamide structure were demonstrated by Muldoon et al. and Spinks et al.;7,33 however, the influence of this factor on the binding affinity is rather unclear. If charge distribution was the dominant force in binding, than it would have to be assumed that true generic antibodies are impossible to generate in vivo.33 However, production of generic antibodies in rabbits and inhibition data obtained in this work suggests that the charge contributions of sulfonamide ligands are unlikely a crucial strength in these interactions. Additionally, Spinks et al.33 hypothesized that the SO2 central group of sulfonamide drugs might represent a dominant recognition point (antigenic subdeterminant) for sulfonamide antibodies generated in vivo. The influence of the SO2NH moiety on antibody specificity was investigated in this study using a p-aminobenzoic acid conjugated to carrier proteins. The antibodies generated against this hapten group did not recognize any of the sulfonamide targets, whereas the presence of the SO2NH grouping in the SA1haptens elicited a generic response of interest to us. It is apparent that this subdeterminant is a necessary structural component of the immunizing hapten group and for the development of binding affinity to sulfonamide drugs. This view was strongly supported by the results of electronic charge distribution analysis carried out for the p-aminobenzoic hapten (carboxylic group was replaced with methyl for calculation) and sulfacetamide molecule. Figure 5 illustrates a considerable charge difference between the two structures, particularly in the aromatic amino group, which is situated at the N4 immunodominant position. It appears from the (33) Spinks, C. A.; Wyatt, G. M.; Lee, H. A.; Morgan, M. R. A. Bioconjugate Chem. 1999, 10, 583-588.

data that changes in partial atomic charge are responsible for binding interaction/competition in these assay systems. Surprisingly enough, when the two-ring (N-sulfanilyl-4-aminobenzoic acid) hapten was used for animal immunization, our laboratory produced good generic antibodies against sulfonamides, whereas other researchers generated rabbit and mice antibodies against the same hapten with only a narrow sulfonamide specificity.1,23,24 The cause of this discrepancy in the cross-reactivity pattern may have been due to factors such as a steric accessibility of conjugated hapten groups for B-lymphocyte (production cells) receptors, the different density of hapten groups in the immunogen, the method of immunization, and other factors. In a previous study, we demonstrated that specificity of the immune response formed at the initial phase of immunization against the steroid hapten was fairly constant during the 183-day immunization period, covering eight immunizations.34 Thus, the stimulation of one population of B-lymphocytes by a hapten at the initial stage of immunization was the determining factor of immunological specificity in the final antibody product. Additionally, the affinity, specificity, and concentration of antibodies raised against small hapten molecules in a polyclonal serum are factors that have a largely individual character, whose course can with defined variability be predicted only for large numbers of animals. Therefore, any generalization of immunochemical data should carefully be considered with respect to the number of immunized animals. From a practical point of view, the natural variability of specificity around the mean can be compensated by immunization of a large number of animals with subsequent selection of the most suitable antibody specificity. Antibody engineering enabled target manipulation of antibody genes resulting in production of generic reagents in vitro. The recombinant mutant, ScFv M.3.4, produced from Mab sulfa 27G3 3,28 allowed the detection of 18 sulfonamides in chicken serum at the MRL level and was found to be a superior reagent in an optical biosensor (Biacore 3000) when compared with other tested sulfonamide binders.35 A direct comparison of the generic pattern of the recombinant mutant ScFv M.3 with our rabbit antibodies is difficult as the inhibition data reported for sulfonamide analytes were obtained using different detection systems and different matrixes. However, three levels of sensitivity toward 19 tested targets appeared in both ELISA systems working with our rabbit antibodies and lanthanide fluoroimmunoassay (LFIA) based on the ScFv M.3. Additionally, different responses toward individual sulfonamides were observed in these assays. For example, the mutant-based LFIA was most sensitive to sulfamethizol (SEZ) and sulfachloropyrazine (SCR) and poorly sensitive to sulfanilamide. Our antibodies 39 and 43 showed a maximum sensitivity to SMX and SP and were the least sensitive for SD and SAM. Relatively low sensitivity to SMZ was indicated in both antibody/assay systems. It is apparent from these results that antibodies originating from immunized animals provided a crossreactivity pattern comparable with that reported for the recombinant mutant. Thus, careful hapten design and hybridoma (34) Franek, M.; Hruska, K. J. Steroid Biochem. Mol. Biol. 1985, 22 (3), 341347. (35) Bienenmann-Ploum, M.; Korpimaki, T.; Haasnoot, W.; Kohen, F. Anal. Chem. Acta 2005, 529, 115-122. (36) Brichta, J.; Hnilova, M.; Viskovic, T. Vet. Med. 2005, 50, 231-252.

technology joint with antibody engineering represent strong means of achieving manipulation of antibodies to generate a broad specificity pattern for sulfonamides. CONCLUSIONS In summary, we have generated polyclonal antibodies exhibiting various degrees of cross-reactivity among commonly used sulfonamide drugs. Most efforts at traditional hapten design have been based on the notion that the immunizing hapten should resemble the analyte as closely as possible. We designed our hapten moieties to mimic half of the sulfonamide molecule. The properties of the formed rabbit antibodies, as shown in Tables 4 and 5, indicated that immunization with the fragment-derived hapten elicited multiselective immune responses and that a superior generic response was raised against the two-ring (Nsulfanilyl-4-aminobenzoic acid) group. Sensitivity of the established generic assays allowed simultaneous detecting all 19 tested targets in buffer and skimmed milk at the level of declared MRL (100 µg‚L-1). The negligible cross-reaction with N4-acetyl metabolites make it possible to measure only responses of parent sulfonamides in the presence of the metabolites. An applicability of the fragmentderived hapten concept to other classes of compounds is possible in the case that a part of the hapten structure is common to all compounds of the group. It should be noted that this structural requirement is well compatible with many groups of food and environmental contaminants including antibiotics and pesticides. A point of the future interest is the production of generic antibodies for sulfonamides providing IC50 responses within a smaller concentration range, e.g., within 1 order of magnitude. An assay based on a generic antibody having less scattered sensitivities could better quantitate sulfonamides in unknown samples at the level of semiquantitative assessment. At present, several advanced immunochemical and recombinant approaches are available providing effective ways to produce antibodies with improved binding properties. With regard to the above discussion, additional immunizations of animals using novel immunogens could lead to more optimal selection of antibody reagents, thus recognizing the actual generic potential of the novel immunogens. An optimal hapten design joined with clonal selection by the antigen in immunized animals as well as surface phage display of the antibodies for the affinity selection (an in vitro analogue of selection by antigen in natural immunity) are key strategies to succeed in achieving improved generic parameters.36 ACKNOWLEDGMENT This work was supported by Grant 525/03/0747 of the Grant Agency of the Czech Republic. The authors thank Z. Zdrahal of the Laboratory of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Brno, for performing the MALDITOF MS analysis and calculation of hapten densities and Z. Kriz of the National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, for performing the molecular modeling and calculation of the charge distribution across the sulfonamide structures. Received for review August 11, 2005. Accepted December 28, 2005. AC0514422 Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

1567