Generic Lanthanide Fluoroimmunoassay for the Simultaneous

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Anal. Chem. 2004, 76, 3091-3098

Generic Lanthanide Fluoroimmunoassay for the Simultaneous Screening of 18 Sulfonamides Using an Engineered Antibody Teemu Korpima 1 ki,*,† Virve Hagren,† Eeva-Christine Brockmann,† and Mika Tuomola‡

Department of Biotechnology, University of Turku, Turku, Finland, and Raisio Life Sciences, Raisio, Finland

Sulfa antibiotics (sulfonamides) are used in veterinary and human medicine for therapeutic and prophylactic purposes. Veterinary use can result in foodstuffs derived from animals being contaminated with residual sulfonamides. Current sulfonamide-screening methods (mainly based on bacterial growth inhibition) are slow and inaccurate, since sensitivities of bacteria to different sulfonamides vary a lot. Therefore, a rapid immunoassay that was able to detect at least 18 different sulfonamides at the MRL level (100 µg/kg) from food samples in a single reaction was developed. The assay was reproducible and adequately accurate for screening purposes. The presence of sulfonamide metabolites did not cause major assay interference. We also demonstrated reliable detection of sulfonamides from a panel of meat, milk, and serum samples with the assay. After their discovery during the 1930s, sulfa antibiotics (sulfonamides) have been used extensively in veterinary and human medicine for therapeutic and prophylactic purposes, being one of the first synthetic chemical agents used to treat bacterial infection. Sulfas all share the p-aminobenzenesulfonamide moiety (Figure 1) and are thus structurally related. As a result of their use in veterinary medicine, meat and milk derived from the medicated animals may contain residual sulfonamides or their inactive metabolites, if proper withdrawal periods are not observed. It has been estimated that ∼5% of the patients on sulfonamide medication receive unwanted symptoms from the drugs,1 so the presence of sulfonamide residues in food is considered potentially harmful for the consumer. Furthermore, it has been shown that low concentrations of antibiotics in the environment may produce genetically altered bacteria resistant to the antibiotics.2 Therefore, a maximum residue limit (MRL) for sulfonamides has been set to 100 µg/kg in the United States and the European Union.3 This creates a need for a simple, low-cost, and fast sulfonamide assay for the screening of a large number of food samples by both the food safety authorities and the producers of animal-derived foods. * Corresponding author: (tel) +358-2-3338685; (fax) +358-2-3338050; (e-mail) [email protected]. † University of Turku. ‡ Raisio Life Sciences. (1) Sheth, H.; Sporns, P. J. Agric. Food Chem. 1991, 39, 1696-1700. (2) Combs, M.; Boyd, S.; Ashraf-Khorassani, M.; Taylor, L. J. Agric. Food Chem. 1997, 45, 1779-1783. (3) Haasnoot, W.; Cazemier, G.; Du Pre, J.; Kemmers-Voncken, A.; BienenmannPloum, M.; Verheijen, R. Food Agric. Immunol. 2000, 12, 15-30. 10.1021/ac049823n CCC: $27.50 Published on Web 05/05/2004

© 2004 American Chemical Society

Although several methods have been developed for the quantification of sulfonamides in foodstuffs using, for example, chromatography,2,4-10 capillaryelectrophoresis,11-13 orimmunoassays,14-21 these methods are slow, expensive, or laborious, may require complex instrumentation, and often detect only a few different sulfonamides. Thus, nowadays, for example, in Finland, the commonly used method of screening for a wide spectrum of different sulfonamides in foodstuffs is bacteriological growth inhibition.22-25 These methods may be relatively low cost but are still quite laborious and slow. Furthermore, growth inhibition assays may produce false positive and negative results as the sensitivity of bacteria to different sulfonamides varies considerably.26 Also, any use of preservatives in, for example, milk samples (4) Abian, J.; Churchwell, M.; Korfmacher, W. J. Chromatogr. 1993, 629, 267276. (5) Cooper, A.; Creaser, C.; Farrington, W.; Tarbin, J.; Shearer, G. Food Addit. Contam. 1995, 12, 167-176. (6) Lin, C.; Hong, C.; Kondo, F. Microbios 1995, 83, 175-183. (7) Volmer, D. Rapid Commun. Mass Spectrom. 1996, 10, 1615-1620. (8) Abjean, J. J. AOAC Int. 1997, 80, 737-740. (9) Le Boulaire, S.; Bauduret, J.-C.; Andre, F. J. Agric. Food Chem. 1997, 45, 2134-2142. (10) Dost, K.; Jones, D.; Davidson, G. Analyst 2000, 125, 1243-1247. (11) Ackermans, M.; Beckers, J.; Everaerts, F.; Hoogland, H.; Tomassen, M. J. Chromatogr. 1992, 596, 101-109. (12) Hows, M.; Perrett, D.; Kay, J. J. Chromatogr. 1997, 768, 97-104. (13) Fuh, M.; Chu, S. Anal. Chim. Acta 2003, 499, 215-221. (14) O’Keeffe, M.; Crabbe, P.; Salden, M.; Wichers, J.; Van Peteghem, C.; Kohen, F.; Pieraccini, G.; Moneti, G. J. Immunol. Methods 2003, 278, 117126. (15) Muldoon, M.; Holtzapple, C.; Deshpande, S.; Beier, R.; Stanker, L. J. Agric. Food Chem. 2000, 48, 537-544. (16) Elliott, C.; Baxter, G.; Crooks, S.; McCaughey, W. Food Agric. Immunol. 1999, 11. (17) Akkoyun, A.; Kohen, V. F.; Bilitewski, U. Sens. Actuators, B 2000, 70, 1218. (18) Lee, N.; Holtzapple, C.; Muldoon, M.; Deshpande, S.; Stanker, L. Food Agric. Immunol. 2001, 13, 5-17. (19) Spinks, C.; Schut, C.; Wyatt, G.; Morgan, M. Food Addit. Contam. 2001, 18, 11-18. (20) Situ, C.; Crooks, S.; Baxter, A.; Ferguson, J.; Elliott, C. Anal. Chim. Acta 2002, 473, 143-149. (21) Walker, C.; Barker, S. J. AOAC Int. 1994, 77, 908-916. (22) Braham, R.; Black, W.; Claxton, J.; Yee, A. J. Food Prot. 2001, 64, 15651573. (23) Koenen-Dierick, K.; Okerman, L.; De Zutter, L.; Degroodt, J.; Van Hoof, J.; Srebrnik, S. Food Addit. Contam. 1995, 12, 77-82. (24) Korsrud, G.; Boison, J.; Nouws, J.; MacNeil, J. J. AOAC. Int. 1998, 81, 21-24. (25) Nouws, J.; Van Egmond, H.; Loeffen, G.; Schouten, J.; Keukens, H.; Smulders, I.; Stegeman, H. Vet. Q. 1999, 21, 21-27. (26) Korsrud, G.; Papich, M.; Fesser, A.; Salisbury, C.; Macneil, J. J. Food Prot. 1996, 59, 161-166.

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Figure 1. Structures and abbreviations of compounds related to sulfanilamide that were used in this study.

can seriously interfere with growth inhibition assays,27 as can matrix effects.28 As compared to growth inhibition assays, immunochemical assays such as lanthanide fluoroimmunoassays (LFIA) have a number of advantages. They are generally much faster, a lot simpler than the growth inhibition assays done on Petri dishes, and if a broad-specificity sulfonamide binder with the capability of recognizing different sulfonamides with similar affinities is used in the assay, the amount of false positives and negatives is drastically smaller. So far, several authors1,3,29-32 have isolated monoclonal antibodies (Mabs) with the capability of binding some sulfonamides with good affinity. Using one of these antibodies, Mab 21C7,32 the detection of eight sulfonamides below the EU MRL level from chicken serum with a surface plasmon resonance (SPR) biosensor has been demonstrated.33 Protein engineering of one of the other existing antibodies, Mab 27G3,3 produced a recombinant antibody M.3.4 capable of detecting, in a LFIA setup, at least 13 different sulfonamides in a buffer system at the MRL level.34-36 Recently, Biacore AB (Uppsala, Sweden) brought to (27) Molina, M.; Althaus, R.; Balasch, S.; Torres, A.; Peris, C.; Fernandez, N. J. Dairy Sci. 2003, 86, 1947-1952. (28) Okerman, L.; de Wasch, K.; van Hoof, J. Analyst 1998, 123, 2361-2365. (29) Muldoon, M.; Font, I.; Beier, R.; Holtzapple, C.; Young, C.; Stanker, L. Food Agric. Immunol. 1999, 11, 117-134. (30) Spinks, C.; Wyatt, G.; Lee, H.; Morgan, M. Bioconjugate Chem. 1999, 10, 583-588. (31) Spinks, C.; Wyatt, G.; Everest, S.; Jackman, R.; Morgan, M. J. Sci. Food Agric. 2002, 82, 428-434. (32) Kohen, F.; Gayer, B.; Amir-Zaltsman, Y.; O’Keeffe, M. Food Agric. Immunol. 2000, 12, 193-201. (33) Haasnoot, W.; Bienenmann-Ploum, M.; Kohen, F. Anal. Chim. Acta 2003, 483, 171-180. (34) 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.

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market SPR biosensor kits (which utilize their own binders) for detection of sulfonamides. In our previous studies,34-36 we engineered antibody M.3.4, which showed great potential as a broad-specificity sulfa binder. In this study, we concentrated on showing that, after optimization of assay parameters, this binder can be used in a LFIA setup for the detection of at least 18 different sulfonamides from real samples such as meat, milk, and serum. We also evaluated assay interference caused by the presence of sulfonamide metabolites and other compounds that might be present in the samples. EXPERIMENTAL SECTION Strains, Plasmids, Reagents, and Instruments. The bacterial host used throughout this work was Escherichia coli K12 strain XL1-Blue (Stratagene, La Jolla, CA). The expression vector used (Figure 2) was constructed from the pAK series of vectors37 in our laboratory. The original pAK vectors were obtained as gifts from the laboratory of Andreas Plu¨ckthun (Biochemisches Institut, Universita¨t Zu¨rich, Switzerland). Recombinant single-chain antibody (scFv) M.3.4 (MW ) 39 000) was engineered in our laboratory34-36 from mouse antibody 27G3A9B10,3 which was a gift from Willem Haasnoot (RIKILT-Institute of Food Safety, Wageningen, The Netherlands). DELFIA Wash Solution, DELFIA Enhancement Solution, and a DELFIA Platewash microtiter plate washer were obtained from Perkin-Elmer Life Sciences (Turku, Finland). The sulfamethazine (35) Korpima¨ki, T.; Rosenberg, J.; Virtanen, P.; Lamminma¨ki, U.; Tuomola, M.; Saviranta, P. Protein Eng. 2003, 16, 37-46. (36) Korpima¨ki, T.; Brockmann, E.-C.; Kuronen, O.; Saraste, M.; Lamminma¨ki, U.; Tuomola, M. J. Agric. Food Chem. 2004, 52, 40-47. (37) Krebber, A.; Bornhauser, S.; Burmester, J.; Honegger, A.; Willuda, J.; Bosshard, H.; Plu ¨ ckthun, A. J. Immunol. Methods 1997, 201, 35-55.

Figure 2. Plasmid expression vector used in the antibody production. A plasmid designed in our laboratory for optimal expression and folding was used as the gene-carrying vector in this work. The E. coli propagation signal (ColE1) and the chloramphenicol resistance selection marker [Cam(R)] were needed for maintaining the plasmid inside the host cells. ScFv antibody (VL-, VH-, and linker domains are separately marked) mRNA transcription was initiated by a lac promoter (lacPO), which was repressed by the lac inhibitor (lacI) until induction with IPTG. The signal sequence (pelB) activated transport of scFv to host periplasmic space for correct folding. The cysteinehexahistidine-tag (Cys(His)6) enabled affinity purification with immobilized metal chelates and site-specific biotinylation with biotinPEO2-maleimide. An insert of E. coli genomic DNA containing two genes (FkpA, SlyX) was included in the plasmid, because this insert has been found to improve scFv folding in vivo.39

derivative conjugated to a long fluorescence lifetime, nine dentate chelate of europium (({2,2′,2′′,2′′′-{[2-(4-isothiocyanatophenyl)ethylimino]bis(methylene)bis{4-{[4-(a-galactopyranoxy)phenyl]ethynyl} pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III)) was synthesized as described previously36 and will be hereafter referred to as Eu-SHZ (MW ) 1704). Streptavidincoated, low-background fluorescence microtitration plates were obtained from Innotrac Diagnostics (Turku, Finland). The antibiotics used in this study were obtained from Sigma-Aldrich (Helsinki, Finland). The N4-acetyl derivatives of various sulfonamides were gifts from Willem Haasnoot. Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Promega (Madison, WI). SB medium was prepared as described in the literature.38 Antifoam 289 and lysozyme were purchased from Sigma-Aldrich, benzonase was bought from Merck (Whitehouse Station, NJ), and biotin-PEO2-maleimide was purchased from Pierce Biotechnology (Rockford, IL). The long-lifetime fluorescence of europium was measured with a Victor 1420 Multilabel Counter (Perkin-Elmer Life Sciences) in a time-resolved mode. ScFv M.3.4 Production, Purification, and Biotinylation. ScFv M.3.4 was produced in a pilot-scale batch fermentation. E. coli cells harboring the expression plasmid pAK3FC-M.3.4 (Figure 2) were precultured in shake flasks containing SB medium with 5 µg/mL tetracyclin and 25 µg/mL chloramphenicol at 26 °C with shaking of 300 rpm. A BioFlo 3000 pilot-scale (4 L) fermentor by New Brunswick Scientific (Edison, NJ) was loaded with 4 L of (38) Sambrook, J.; Fritch, E.; Maniatis, T. Molecular cloning: A laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989.

sterile SB medium fortified with 5 µg/mL tetracyclin, 25 µg/mL chloramphenicol, 0.1% glucose, 2 mM MgSO4, and 100 µL of antifoam 289 and inoculated to an optical density (OD600 nm) of 0.001 with precultured cells. The fermentor was set to maintain a temperature of 26 °C, an airflow of 4 L/min through the sparger, and an agitation rate of 280 rpm. A control loop was set up so that the agitation rate was automatically increased if the relative dissolved oxygen content in the fermentation medium (DO%) dropped below 30%. Cell culturing was continued, until the cells reached an OD600 nm of 3 and the scFv expression was induced with the addition of IPTG to a concentration of 400 µM. Fermentation was continued for a further 6 h after which the cells were collected by centrifugation (7000g, 15 min) and frozen. Purification of scFv M.3.4 was done using periplasmic extraction, cation-exchange chromatography, and immobilized metal affinity chromatography (IMAC). Site-specific biotinylation of scFv M.3.4 was done while the antibody remained bound in the IMAC column. The frozen E. coli cells were thawed on ice and weighed. The cells were quickly suspended in 5 mL of warm (25 °C) lysis buffer (15 mM Tris pH 8, 4 mM EDTA, 10 mg/L lysozyme) per gram of cell wet weight followed by the addition of 10 mL of ice cold (4 °C) lysis buffer per gram of cell wet weight. After thorough mixing, nucleic acids (although only the periplasm of most cells is lysed, some cells lyse completely) were precipitated by slow addition of 1.3 mL of 1% polyethylenimine per gram of cell wet weight. The precipitated nucleic acids were removed by centrifugation (11000g, 10 min, 4 °C). To the supernatant, 30 units of benzonase per gram of cell wet weight was added and the pH of the solution was adjusted roughly to 6 using 1 M acetic acid and pH paper. Finally, the lysate was diluted with water until the conductance of the solution was under 2.5 mS/cm. The adapter of a Streamline 15 expanded bed chromatography column (Amersham Biosciences, Uppsala, Sweden) loaded with 75 mL of Streamline SP cation-exchange matrix (Amersham Biosciences) was set to the 55-cm mark and equilibrated with 500 mL of running buffer (30 mM MES pH 6, 5 mM NaCl) in an upward direction. The cleared lysate was pumped through the column in an upward direction. The adapter was lowered to the 45-cm mark, and the column was washed in an upward direction with 1000 mL of running buffer. The chromatography matrix was allowed to settle down for 10 min, before the adapter was lowered to the 17-cm mark and washed in a downward direction with 200 mL of running buffer. Bound proteins were then eluted in a downward direction with 200 mL of elution buffer (30 mM MES pH 6, 250 mM NaCl) collecting 11-mL fractions. Protein content of the fractions was estimated spectrophotometrically (A280 nm). The fractions containing protein were pooled. The pH of the fraction pool was adjusted to 7.4 with 1 M NaOH followed by the addition of dithiothreitol (DTT) to a final concentration of 1 mM. The solution was incubated at room temperature with gentle stirring for 1 h so that DTT had time to reduce the cysteine residues in the tails of the scFvs. A column packed with 2 mL of Ni-NTA Superflow IMAC matrix (Qiagen, Hilden, Germany) was equilibrated with 10 mL of running buffer (50 mM phosphate pH 7, 0.3 M NaCl, 0.02% Tween 20). Next, the sample was passed through the column. The column was washed with 6 mL of running buffer followed by 4 mL of running buffer containing 100 µM biotin-PEO2-maleimide. The column was Analytical Chemistry, Vol. 76, No. 11, June 1, 2004

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allowed to stand for 1.5 h at room temperature in order for the site-specific biotinylation of the CysHis6 tails to take place. The column was then further washed with 6 mL of running buffer. Bound proteins were eluted with 10 mL of elution buffer (50 mM phosphate pH 7, 0.3 M NaCl, 0.02% Tween 20, 500 mM imidazole), collecting 2-mL fractions. Protein content of the fractions was estimated spectrophotometrically, and the protein-rich fractions were pooled. The scFv solution was concentrated to a 0.5-mL volume using a Centricon YM-10 ultrafiltration device (Millipore, Billerica, MA). The buffer of the scFv preparation was switched to TSAT (50 mM Tris pH 7.75, 0.9% NaCl, 0.05% NaN3, 0.02% Tween 20) with a NAP-5 gel filtration column (Amersham Biosciences). The final protein concentration was determined spectrophotometrically. Competitive Lanthanide Fluoroimmunoassay for Sulfonamides. The competitive lanthanide fluoroimmunoassays were generally performed as follows. The assay buffer that was used for all dilutions was 100 mM phosphate buffer, pH 6.5 (pH 7.75 was used before pH optimization experiments) with 0.9% NaCl, 0.01% Tween 40, and 0.01% casein. Streptavidin-coated microtitration plates were washed once with DELFIA Wash Solution. Duplicate (five replicates were used for the SHZ standard curve) 100-µL aliquots of each sample and each sulfonamide standard in assay buffer were added to the wells. Next, a 50-µL aliquot of 24 ng/mL Eu-SHZ (this was varied during tracer concentration optimization) in assay buffer and a 50-µL aliquot of 800 ng/mL Bio-scFv M.3.4 (varied during antibody concentration optimization) in assay buffer was added to each well. The plates were incubated for 15 min (30 min was used before incubation time optimization) at room temperature with gentle shaking and washed four times as above. A 200-µL aliquot of enhancement solution was added to each well. After 15-min incubation with gentle shaking, the long-lifetime fluorescence of europium was measured. The B/B0 ratios for each sample and standard were calculated using the formula B/B0 ) S/S0, where S is the fluorescence signal of the given sample or standard and S0 is the fluorescence at zero dose of the analyte. Background fluorescence caused by nonspecific binding (nsb) of the tracer was not subtracted from the signals as it was insignificantly small. For the determination of the IC50 concentrations (the concentration of analyte needed to inhibit 50% of tracer binding in the assay) of the sulfonamide analytes, six different concentrations of each analyte were used. Sigmoidal curves were fitted to the data using the Sigmoidal inhibition from B0 to nsb model of LSW Data Analysis Toolbox ver 1.1.1 (MDL Information Systems, San Leandro, CA) for the calculation of the IC50 concentrations. Nine different concentrations were measured for the compilation of the SHZ standard curve, and the sigmoidal curve was fitted as above. Limits of detection for different sample matrixes were obtained, as is usually done, by multiplying the standard deviation of the S0 of 20 analyte free samples by 3, subtracting it from the average S0, and converting the result to a concentration with the standard curve. Sample Preparation. Twenty batches of Finnish bovine tenderloin were purchased from local shops. Twenty samples of bovine whole milk (each from a different cow) were obtained from a local organic farm. Twenty samples of chicken serum (each from 3094

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a different chicken) were obtained from a local slaughterhouse. All samples were assumed to be sulfonamide-free. Each beef sample was weighed, and 1 mL of assay buffer was added per gram. All of the sulfonamide spikings were done at this point. The samples were homogenized with an Ultra Turrax T-25 homogenizer (IKA Werke, Staufen, Germany). After centrifugation (13000g, 5 min), trichloroacetic acid (TCA, 25% aqueous solution) was added to the supernatants to a final concentration of 1.4% in order to precipitate most of the proteins. Samples were centifugated (13000g, 5 min) again, and 1/15 volume of neutralization buffer (460 mM glycine, 540 mM NaOH) was mixed to the supernatants. The resulting solutions were used directly in the competitive sulfonamide LFIA as samples. To each sample of whole milk, TCA was added to a final concentration of 2.3% in order to precipitate most of the milk proteins. Samples were then centrifugated (13000g, 5 min), and 1/7 volume of neutralization buffer was mixed to the supernatants. The resulting solutions were diluted 1/5 in the assay buffer and used in the LFIA as samples. To each sample of serum, TCA was added to the final concentration of 2.3% so that most of the serum proteins were precipitated. Samples were centrifugated (13000g, 5 min), and 1/7 volume of neutralization buffer was mixed to the supernatants. The resulting solutions were diluted 1/4 in the assay buffer for the LFIA. RESULTS ScFv M.3.4 Production, Purification, and Biotinylation. For antibody production purposes, the gene of scFv M.3.436 was cloned as a Cys-His6-tail fusion to a pAK3FC antibody expression vector (Figure 2), and the vector harboring E. coli cells was cultivated in a pilot-scale batch fermentation reaction. Plasmid pAK3FC has an insert of E. coli genomic DNA, which contains two genes (FkpA, SlyX) that have been experimentally found to improve scFv folding in vivo.39 The plasmid also has a pelB signal sequence, which directs the antibodies to E. coli periplasmic space for folding. By using a low culture temperature (26 °C), we sought to obtain a good antibody-folding efficiency and by using only a moderate inducer concentration (400 µM IPTG) we tried not to overload the periplasmic spaces of the expressing cells with unfolded or folding antibodies in order to avoid aggregate formation. Purification was started with a mild chemical lysis in order to release only the contents of the periplasmic spaces to the supernatant. This way the native cytosolic proteins of E. coli did not contaminate the lysate and complicate the following purification, which was started with the collection of scFv M.3.4 from the supernatant using expanded bed cation-exchange chromatography in a low pH where M.3.4 is positively charged. Expanded bed columns do not easily become clogged and rescue recombinant antibodies with good efficiencies, so they are best used as the first purification step. After reduction of the cysteine residues of the antibody CysHis6 tails with DTT, an IMAC column was used to capture the scFv M.3.4 molecules by the hexahistidine part of the tails. M.3.4 molecules bound to the column were then site-specifically biotinylated with biotin-PEO2-maleimide targeting the free cysteine (39) Bothmann, H.; Plu ¨ ckthun, A. J. Biol. Chem. 2000, 275, 17100-17105.

Figure 3. Optimization of competitive assay tracer and binder concentrations. Illustrated in the figure is the relative assay signal intensity (B/B0) given by the presence of 100 µg/L SHZ in the assay reaction as the function of the competitive assay tracer Eu-SHZ (b) or binder Bio-scFv M.3.4 (×) concentrations. The concentration of Eu-SHZ was 3.5 nM in the experiment where the binder concentration was varied whereas Bio-scFv M.3.4 concentration was 5.1 nM in the experiment where the tracer concentration was varied.

residues of the Cys-His6 tails. In-column biotinylation made it possible to stringently wash away unreacted biotin before elution of the antibodies from the column. The final yield of Bio-M.3.4 was ∼0.5 mg/L of fermentation volume, which means that we produced enough immunoreagent for ∼50 000 assays with a simple pilot-scale batch production run. If more complex high cell density cultivations were to be used,40 antibody yield could probably be pushed significantly upward without the need for higher volumes. Competitive Lanthanide Fluoroimmunoassay Optimization. The concentrations of the tracer (Eu-SHZ) and the binder (Bio-scFv M.3.4) in the LFIA reaction were optimized in experiments where these two parameters were systematically varied. For each tracer and binder concentration, europium fluorescence at zero dose of analyte (S0) and relative assay signal (B/B0) in the presence of 100 µg/L SHZ in the assay were determined. The lower the B/B0 given by the fixed amount of analyte (100 µg/L SHZ), the more sensitive the assay could be assumed to be with those binder and tracer concentrations. The results of these experiments are shown in Figure 3. Based on the results, EuSHZ and Bio-scFv M.3.4 concentrations to be used in the assay were selected to be 6 (3.5 nM) and 200 ng/mL (5.1 nM), respectively. With these concentrations, optimal sensitivity was obtained while retaining a good S0 of hundreds of thousands of fluorescence counts. To pick the optimal pH for the assay buffer, an experiment was done where different assay pHs were tested. For each pH, both S0 and B/B0 given by 25 µg/L SHZ were measured (Figure 4). After a review of the results, it was decided that assay buffer pH 6.5 would give the best combination of S0 and sensitivity. The impact of incubation time on the assay performance was also assessed. For each incubation time, S0 was measured (Figure (40) Horn, U.; Strittmatter, W.; Krebber, A.; Knupfer, U.; Kujau, M.; Wenderoth, R.; Muller, K.; Matzku, S.; Plu ¨ ckthun, A.; Riesenberg, D. Appl. Microbiol. Biot. 1996, 46, 524-532.

Figure 4. Optimization of assay buffer pH. Shown in the figure are the assay europium fluorescence signal (S0) at the zero dose of analyte (b) and the relative assay signal intensity (B/B0) at the presence of 25 µg/L of SHZ in the assay reaction (×) as the function of assay buffer pH. Cts is fluorescence counts.

Figure 5. Optimization of assay incubation time. Shown in the figure is the assay europium fluorescence signal (S0) at the zero dose of analyte as the function of assay incubation time. Cts is fluorescence counts.

5). The results showed that the assay signal reached 95% of equilibrium signal in ∼15 min, and so this incubation time was selected for the final assay. Using the optimized assay parameters, a standard curve for sulfamethazine was established using nine different standard concentrations of SHZ (Figure 6). The signal coefficients of variation (CV) were under 5% for each datapoint. The fit of a sigmoidal curve to the obtained data was good with r2 value of 1.00. The assay precision profile showed good accuracy (CV 1000 20 13 >1000 180 300 11 170000 >100000 >100000 >100000 >100000

a Abbreviations not defined in Figure 1: N4-acetyl (AC-), tetracycline (TET), chloramphenicol (CM), kanamycin (KAN), and ampicillin (AMP).

the sulfonamide-related drug dapsone with a better sensitivity than SHZ. The N4-acetyl metabolites of sulfonamides were generally detected with ∼25-fold poorer sensitivity than the corresponding parent compounds. The IC50 values for sulfa antibiotic SAN, prodrug SAZ, the tested nonsulfonamide antibiotics, and paminobenzoic acid (PABA), which can be found in some foodstuffs, were all higher than 1000 µg/L, so they can be considered undetectable with the assay. Meat, Milk, and Serum Samples. The optimized LFIA for sulfonamide detection was tested with three different sample matrixes: bovine meat, bovine whole milk, and chicken serum. For each sample matrix type, 20 sulfonamide-free samples and 20 samples fortified with 100 µg/kg SHZ were analyzed with the assay (Table 2). The standard deviation of the signal of the sulfonamide-free samples multiplied by 3 was used to determine 3096 Analytical Chemistry, Vol. 76, No. 11, June 1, 2004

the limit of detection (LOD), which did not vary much between sample types. The average recovery, however, significantly varied between different sample types, being as low as 25% for meat and as high as 79% for whole milk. Thus, the LOD in sample (taking into account average recovery and sample dilution) was the lowest for milk (16 µg/L) and highest for meat (29 µg/kg). The withinrun precision was worst (24%) for meat and under 10% for milk and serum. To assess the between-run precision of the assay, five samples of chicken serum were analyzed three times (Table 2). Based on this experiment, between-run precision was not significantly worse than within-run precision. The between-run variation of the assay signals given by the sulfonamide-free samples was quite high (16%), but this was probably caused by small variations in assay conditions (e.g., tracer and antibody dilutions, incubation time, and temperature) and did not seem to affect assay results. DISCUSSION Competitive Lanthanide Fluoroimmunoassay for Sulfonamides. In the setup of the competitive LFIA, the first parameters to be optimized were the tracer and binder concentrations in the assay reaction. We found out that if both tracer and binder concentrations were slightly smaller than the dissociation constant (Kd) of the antibody (for scFv M.3.4, Kd,SHZ,pH 7.75 ) 20 nM), assay sensitivity was optimal, as could be expected based on our mathematical models of competitive immunoassays, while specific fluorescence signal at zero dose of analyte (S0) was still good. It is interesting to note that, if antibodies of very high affinities are used in competitive assays, the specific activity of the label becomes an important factor contributing to S0 staying measurable at the optimal concentration of tracer and antibody. In those kinds of situations, lanthanide chelate labels might be better than traditional ELISA labels because of the high specific activity of these labels. However, this was not the case with M.3.4, which can be said to have only a moderate affinity and thus even simple ELISA labels would be sufficient for obtaining signal. We assumed that assay reaction pH would have an effect on sulfonamide binding by our antibody, since pH affects the protonation state of the amino acid residues that form the binding site of M.3.4 and the protonation states of the analyte sulfonamide and the tracer sulfonamide derivative, since sulfas are amphoteric in nature. Our results showed that, at pH 6.5, both the sensitivity of the assay for SHZ and assay S0 were the highest. At this pH, different sulfas are either neutral or negatively charged (SHZ pKa ) 7.4 and SMT pKa ) 5.45, for example). However, there does not seem to be any general relationship between the pKa of a sulfa and the sensitivity of the LFIA toward it as shown by the crossreactivity measurements (Table 1). We conclude that M.3.4 might bind the ionic form of a sulfa with better affinity than the neutral form or vice versa, but wrong protonation state does not apparently exclude binding. The incubation time optimization experiments (Figure 5) showed that the LFIA reached 95% of equilibrium signal in 15 min. We chose 15-min incubation time for our assay runs, since we were performing the immunoassays manually and did not want variation (as compared to the total incubation time) to be introduced to the incubation times of different wells by the manual pipetting process. However, since the assay reached 83% saturation in 5 min, it might be possible to reduce the incubation time to 5

Table 2. Assay Performance Data bovine meat (n ) 20) blank signal (S0) variation within-run LOD (SHZ) average recovery (100 µg/L SHZ) LOD in sample (SHZ) within-run precision (100 µg/L SHZ)

4.3% CV 1.8 µg/L 25% 29 µg/kg 24% CV

bovine whole milk (n ) 20)

chicken serum (n ) 20)

3.1% CV 1.2 µg/L 79% 16 µg/L 9.3% CV

2.8% CV 1.1 µg/L 36% 25 µg/L 7.8% CV

chicken serum (n ) 5, 3 runs) blank signal (S0) variation between-run between-run precision (100 µg/L SHZ)

16% CV 8.0% CV

min if automation in assay execution would be used. Furthermore, by using surface readout of lanthanide fluorescence (the 9d-Eu chelate we used is intrinsically fluorescent), the enhancement step could be omitted, thereby making the assay even faster. The final calibration curve for SHZ using the optimized LFIA parameters (Figure 6) showed excellent fit to the sigmoidal model function we used. The assay precision profile showed that the assay was rather accurate (concentration CV < 10%) over a range of 2-3 orders of magnitude. This means that if the identity of the contaminating sulfonamide was clear so that correct calibrator compound could be selected, our assay could probably be used, in addition to screening purposes, for rough concentration measurements as well. Cross-reactivity studies showed that the described sulfonamide LFIA was capable of detecting at least 18 different sulfonamides with limits of detection lower than the MRL level. There was also one drug, DAP, which is not a sulfonamide but a structurally related compound, that the assay detected with about the same sensitivity as SHZ. The IC50 values for most of the sulfas were within 2 orders of magnitude of each other. Thus, there is the possibility of false positives if samples are contaminated with concentrations of STZ just below the MRL, for example, just as in the case of microbiological inhibition assays, but the danger of false negatives is minimal as opposed to these assays. In screening assays, false positives are much less dangerous than false negatives, since positive samples should nevertheless always be reevaluated using other analytical methods such as HPLC. In the end, the final cutoff signal of the assay should be decided according to the need of the user, i.e., which sulfas need to be screened for. Sulfonamide prodrug SAZ, which is cleaved to SPY in vivo, was not detected by our assay with adequate sensitivity for screening, probably due to the bulky group attached to the N4 position of SAZ. Luckily, this sulfa can still be indirectly detected due to the conversion to SPY, which the assay is sensitive for. Therefore, the only tested sulfonamide that was not detected with an adequate sensitivity by the LFIA was SAN. The poor binding of SAN by M.3.4 is probably caused by the lack of N1 substitution in the structure of this sulfa. Sulfonamides are metabolized in vivo to their N4-acetylated forms. This form is inactive, so it should not be detected by screening assays. According to our measurements, it seems that M.3.4 generally binds these metabolites with ∼25-fold poorer affinity than the corresponding parent compound. Still, the presence of metabolites of some sulfas that the competitive assay is highly sensitive to, such as STZ, might cause some false

positives, since the LFIA is about equally sensitive to SHZ and AC-STZ, for example. Finally, the presence of PABA (which sulfonamides structurally mimic in the competitive inhibition of dihydropteroate synthase) or antibiotics structurally unrelated to sulfonamides in samples should not cause any problems, since the competitive LFIA did not react to these compounds. Assay Performance with Meat, Milk, and Serum Samples. To assess the usefullness of the competitive sulfonamide LFIA, assay performance with three different sample materials was evaluated. Bovine meat, bovine whole milk, and chicken serum samples were spiked with MRL concentration of SHZ and measured with the LFIA. Since SHZ was the sulfa that the assay was least sensitive to, it was assumed that if SHZ can be consistently detected, then the rest of the sulfas should also be detectable at the MRL. According to the results obtained, SHZ can be reliably detected from all three sample matrixes, even though the recoveries from serum and meat were quite low. The recovery problems might have been caused by adsorption of the analyte on the sample proteins and it might be possible to solve these problems with the use of, for example, some methanol for sample extraction.19 The within-run precision of the assay was more than adequate for a screening assay in our opinion, even in the case of the meat samples. Between-run precision was tested only for the serum samples, and it was not significantly worse than the within-run precision. S0 signal variation was higher between-run than within-run. This was probably due to small differences caused by tracer and antibody dilution preparation and small variations in assay incubation time and temperature, which can in the case of competitive assays significantly affect S0. This variation did not seem to affect assay results, however. CONCLUSIONS A broad-specificity sulfonamide antibody (M.3.4) was used in this study as the binder of a competitive sulfonamide screening assay utilizing LFIA technology. The developed LFIA is very fast, is capable of detecting at least 18 different sulfonamides below the MRL concentration, and does not display major interference from sulfonamide metabolites or other antibiotics that might be present in food samples. We also demonstrated that sulfonamides in meat, milk, and serum can be screened with the described assay without major matrix effects. ACKNOWLEDGMENT This work has been supported by a grant from the Finnish National Technology Agency (TEKES). We thank the organic farm in Kuralan Kyla¨ma¨ki, Turku, Finland, for kindly providing Analytical Chemistry, Vol. 76, No. 11, June 1, 2004

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the whole milk samples used in this study and Willem Haasnoot (RIKILT DLO, Wageningen, The Netherlands) for providing the N4-acetyl derivatives of sulfonamides. We also thank Markus Vehnia¨inen for constructing plasmid pAK3FC.

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Received for review January 30, 2004. Accepted March 30, 2004. AC049823N